Polynucleotides encoding an actriib antibody

ABSTRACT

The disclosure provides, among other aspects, neutralizing antibodies and portions thereof that bind to ActRIIB and uses for same.

RELATED APPLICATION

This application is a continuation of and claims priority to U.S.application Ser. No. 13/283,552, filed Oct. 27, 2011. The specificationsof each of the foregoing applications are incorporated herein byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 7, 2014, isnamed PHPH064103_Seq.txt and is 26,546 bytes in size.

BACKGROUND OF THE INVENTION

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. The superfamilyincludes proteins that are variously described as Growth andDifferentiation Factors (GDFs), Bone Morphogenetic Proteins (BMPs),activins and inhibins.

By manipulating the activity of a member of the TGF-beta superfamily, itis often possible to cause significant physiological changes in anorganism. For example, GDF8 (myostatin) is a well-known regulator ofskeletal muscle mass and strength. The Piedmontese and Belgian Bluecattle breeds carry a loss-of-function mutation in the myostatin genethat causes a marked increase in muscle mass. Grobet et al., Nat Genet.1997, 17(1):71-4. Furthermore, in humans, inactive alleles of GDF8 areassociated with increased muscle mass and, reportedly, exceptionalstrength. Schuelke et al., N Engl J Med 2004, 350:2682-8. Other membersof the superfamily, such as activin A, are also reported to regulateskeletal muscle. Modulators of GDF8 and activin are in clinicaldevelopment for the treatment of muscle conditions. Antibodies directedto myostatin promote muscle formation in animal models, although resultsin human clinical trials have not demonstrated prominent increases inskeletal muscle mass or strength, raising a question as to whether othermembers of the TGF-beta superfamily may regulate muscle growth. Walkeret al., Ann Neurol. 2008, 63:561-71. The activin receptor type IIB(ActRIIB, also known as ACVR2B), is a high affinity receptor formyostatin, activin A and other members of the TGF-beta superfamily,including GDF11 and other activins. Lee et al. Proc Natl Acad Sci USA2005, 102:18117-22; Mathews et al. Science 1992 255:1702-5; WO 00/43781;WO 2006/012627. An ActRIIB-Fc fusion protein acts as a high affinityantagonist to each of these ligands and promotes substantial musclegrowth in animal models as well as humans. Lee et al. Proc Natl Acad SciUSA 2005, 102:18117-22; WO 2004/039948; WO 2006/012627; WO 2008/097541.ActRIIB-Fc is also known to promote bone formation and, in some cases,affect other tissues. WO 95/10611; Hamrick et al. Calcif Tissue Int2002, 71:63-68; WO 2006/012627; WO 2008/097541. Similarly, it has beenproposed that antibodies that bind to ActRIIB and disrupt ligand bindingand/or signaling (e.g., neutralizing antibodies) can be used to promotemuscle or bone formation and treat a variety of disorders. U.S. Pat. No.6,656,475; WO 2006/012627; WO 2008/097541.

Thus, it is an object of this disclosure to provide antibodies that bindto ActRIIB and uses for same.

SUMMARY OF THE INVENTION

The disclosure provides, among other aspects, antibodies and fragmentsthereof that bind to ActRIIB and inhibit ActRIIB-mediated signaling. Avariety of uses for such proteins are described herein. For example, theantibodies may be used to treat a variety of diseases, includingdisorders of skeletal muscle and bone, and as part of assays to identifyknown and novel ActRIIB-binding agents. In a further embodiment,antibodies that bind to ActRIIB and inhibit ligand binding may be usedin assays to detect and characterize antibodies that may be generated ina human in response to administration of a polypeptide comprising a partor all of the extracellular domain of ActRIIB, such as an ActRIIB-Fcfusion protein.

In certain embodiments, the disclosure relates to antibodies, andfragments of antibodies (e.g., Fab, scFv) that specifically bind toActRIIB. The binding agents can be characterized by their ability toinhibit binding to or signaling through ActRIIB by one or more ligands,such as myostatin, GDF11, activin A, activin B or others described inthe art. Binding agents may cross-block the binding of at least oneantibody disclosed herein, such as Ab-17G05, to ActRIIB and/or to becross-blocked from binding ActRIIB by at least one of said antibodies.In certain aspects, the anti-ActRIIB antibody is a therapeutic antibodyor functional fragment thereof. An anti-ActRIIB antibody or functionalfragment thereof may bind to a ligand-binding domain of ActRIIB, theboundaries and attributes of which are described herein. An anti-ActRIIBantibody or functional fragment thereof may bind to ActRIIB betweenamino acids 19-134 of SEQ ID NO: 1. Any of the antibodies and fragmentsdescribed herein may bind to ActRIIB with a KD (dissociation constant)of 1 nM, 100 pM, 50 pM, 20 pM, 10 pM or less. An anti-ActRIIB antibodymay inhibit the binding of one or more ligands to ActRIIB, includingmyostatin, activin A, GDF11, activin B, BMP9 or BMP10. Because bothactivins and myostatin, and possibly GDF11, act as negative regulatorsof skeletal muscle mass, an anti-ActRIIB antibody may be selected so asto inhibit the binding of two of more of the aforementioned ligands toActRIIB. An anti-ActRIIB antibody may inhibit the signaling caused by anActRIIB ligand, such as myostatin, and such signaling may be measured bya Smad dependent reporter gene assay, such as the A204 assay describedin the Examples. Smad activation may also be assessed by measuring thelevels of phosphor-Smads, particularly Smad2 or Smad3. In certaininstances, it will be desirable to selectively bind ActRIIB with littleor no binding to the related receptor ActRIIA, and accordingly, ananti-ActRIIB antibody or functional fragment thereof may bind to ActRIIBwith a 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold or1000-fold or greater/better affinity than it binds to ActRIIA. Ananti-ActRIIB antibody or functional fragment thereof may be of any ofthe known immunoglobulin isotypes, and particularly IgG1, IgG2 or IgG4,and may have an altered effector function. An altered effector functionmay be achieved by modifying or mutating the Fc region, and this may beperformed to create an antibody having reduced ADCC or CDC reactivity.An anti-ActRIIB antibody or fragment thereof may promote skeletal musclegrowth in vivo, particularly in a mouse, non-human primate or a human.

An anti-ActRIIB antibody or fragment thereof may comprise at least oneCDR sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to a CDR selected from the groupconsisting of SEQ ID NOs: 37-42. The antibody or fragment thereof maycomprise at least two, three, four, five or six of the foregoing CDRsequences and may, for example, comprise three CDRs, CDR-H1, CDR-H2, andCDR-H3 wherein CDR-H1 comprises a sequence that is at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 37, CDR-H2 comprises a sequence that is at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 38, and CDR-H3 comprises a sequence that is at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identicalto SEQ ID NO: 39. The antibody or fragment thereof may comprise threeCDRs, CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 comprises a sequencethat is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identical to SEQ ID NO: 40, CDR-H2 comprises a sequence thatis at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 41, and CDR-H3 comprises a sequence thatis at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 42. Other combinations and permutationsof the foregoing CDR sequences and variants are included within thedisclosure. An anti-ActRIIB antibody may comprise a heavy chain whereinsaid heavy chain comprises a polypeptide having at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to thesequence given in SEQ ID NO: 15. An anti-ActRIIB antibody may comprise alight chain wherein said light chain comprises a polypeptide having atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity to the sequence given in SEQ ID NO: 16. An anti-ActRIIBantibody may comprise any combination of the foregoing heavy chain andlight chain sequences.

The anti-ActRIIB antibodies and functional fragments described may beformulated as pharmaceutical compositions comprising such antibody orfunctional fragment. A pharmaceutical composition may comprise apharmaceutically acceptable diluent or carrier.

In certain aspects the disclosure provides isolated polynucleotidesequences encoding the antibody or functional fragment thereof describedherein, including any of the polynucleotides encoding each of the heavyand light chains described, as well as the variable domains and therespective CDR portions. The disclosure further provides cloning orexpression vectors comprising any of the foregoing isolatedpolynucleotide sequences, and cells, particularly host cells such as CHOor NSO cells comprising any of the above nucleic acids or vectors. Suchhost cells may be used to produce the anti-ActRIIB antibodies described.In certain aspects the disclosure provides processes for the productionof an antibody or functional fragment thereof described herein,comprising culturing a host cell comprising a cloning or expressionvector encoding the antibody or functional fragment thereof, andisolating the antibody or functional fragment thereof.

A variety of uses for binding agents that neutralize ActRIIB have beendescribed, and thus in certain aspects, the disclosure provides methodsfor using anti-ActRIIB antibodies or functional fragments. For example,such agents, and pharmaceutical preparations containing same, may beused in a method of treating a patient suffering from a musculoskeletaldisease or disorder; acute and/or chronic renal disease or failure;cancer; breast cancer; Parkinson's Disease; conditions associated withneuronal death; ALS; brain atrophy; dementia; anemia; liver, kidney andpulmonary fibrosis; one or more age-related condition; rhabdomyosarcoma;bone-loss inducing cancer. Such methods may comprise the step of:administering an effective dose of an antibody disclosed herein orfunctional fragment thereof to said patient. Examples of musculoskeletaldiseases or disorders include muscle atrophy, myopathy, myotonia, acongential myopathy, nemalene myopathy, multi/minicore myopathy andmyotubular (centronuclear) myopathy, mitochondrial myopathy, familialperiodic paralysis, inflammatory myopathy, metabolic myopathy, aglycogen or lipid storage disease, dermatomyositisis, polymyositis,inclusion body myositis, myositis ossificans, rhabdomyolysis andmyoglobinurias; a dystrophy, including Duchenne, Becker, myotonic,fascioscapulohumeral, Emery-Dreifuss, oculopharyngeal, scapulohumeral,limb girdle, Fukuyama, a congenital muscular dystrophy, or hereditarydistal myopathy; osteoporosis; a bone fracture; short stature; dwarfism;prolonged bed rest; voluntary inactivity; and/or involuntary inactivity.As further examples, a patient being treated may be elderly, may havespent time in a zero gravity environment or may have undergone a periodof inactivity, and treatment may be initiated prior to theaforementioned event. Such a patient may have a fracture to a limb orjoint or have undergone or be about to undergo hip or knee replacementsurgery.

In certain aspects, the disclosure provides methods for usinganti-ActRIIB antibodies to detect ActRIIB in cells and tissues, and alsoin assays designed to detect or assess other antibodies that bind toActRIIB. Reagents that include the ligand-binding portion of ActRIIB(e.g., an ActRIIB-Fc fusion protein) are in development as therapeuticagents, and as with all biologic products, it is of interest todetermine whether such agents cause the production in patients ofantibodies against the therapeutic protein and whether such antibodiesare neutralizing. Accordingly, a method described herein for detectingor characterizing anti-ActRIIB antibodies in blood may comprise a stepof contacting an ActRIIB polypeptide (e.g. a polypeptide comprising anActRIIB ligand binding domain) with a neutralizing anti-ActRIIBantibody. In an embodiment, such a method may comprise (i) forming amixture comprising a sample (e.g., a blood or serum sample from apatient treated with an ActRIIB-Fc fusion protein or a placebo), anActRIIB polypeptide and a control antibody that is a known neutralizinganti-ActRIIB antibody; and (ii) measuring the amount of control antibodythat is bound to the ActRIIB polypeptide, wherein the ActRIIBpolypeptide is a polypeptide comprising a ligand binding domain ofActRIIB. A decrease in the amount of control antibody bound to theActRIIB polypeptide relative to a standard indicates that the samplecontains a neutralizing anti-ActRIIB antibody. The standard may be amixture comprising the ActRIIB polypeptide and the control antibody. Thestandard may further comprise a sample that is known to contain nosubstantial amount of neutralizing anti-ActRIIB antibody, and mayfurther comprise a sample that contains a known amount of neutralizinganti-ActRIIB antibody. In another format, the assay may comprise (i)forming a mixture comprising the sample, an ActRIIB polypeptide and aligand that binds to ActRIIB; (ii) measuring the amount of ligand thatis bound to the ActRIIB polypeptide and comparing the amount of ligandthat is bound with a standard, wherein the standard is measured amountof ligand bound to the ActRIIB polypeptide in a mixture comprising theActRIIB polypeptide, the ligand and a control antibody that is a knownneutralizing anti-ActRIIB antibody, and wherein the ActRIIB polypeptideis a polypeptide comprising a ligand binding domain of ActRIIB. Acomparison of the amount of ligand bound to the ActRIIB polypeptide inthe mixture versus the amount of ligand bound to the ActRIIB polypeptidein the standard may be used to assess the presence or absence of aneutralizing anti-ActRIIB antibody in the sample, with decreased ligandbinding indicating higher levels of neutralizing anti-ActRIIB antibody.The ligand may be any known ligand for ActRIIB and may be selected fromthe group consisting of: activin A, activin B, myostatin and GDF11. Ineach assay format, the sample may contain blood or a blood product,optionally from a patient treated with an ActRIIB-Fc fusion protein or apatient that has not been treated with an ActRIIB-Fc fusion protein. Theneutralizing anti-ActRIIB antibody may be any of the anti-ActRIIBantibodies disclosed herein and may, for example comprise at least oneCDR sequence having at least 80% identity to a CDR selected from thegroup consisting of SEQ ID NOs: 37-42 or may be the Ab-17G05.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entireties as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 depicts kinetic characterization of Fab-17G05 binding tohActRIIB-hFc as determined by BIACORE™-based analysis at 25° C. (A) or37° C. (B). hActRIIB-hFc was captured on a chip with covalentlyimmobilized antibody against human IgG1 Fc and then exposed to Fab-17G05at concentrations ranging from 0.0195 μg/ml to 5 μg/ml. RU, responseunits. As determined by nonlinear regression, the K_(D) was 5.5×10⁻¹⁰ at25° C. and 1.9×10⁻⁹ at 37° C.

FIG. 2 depicts kinetic characterization of Ab-17G05 binding tohActRIIB-mFc as determined by BIACORE™-based analysis at 25° C. (A) or37° C. (B). hActRIIB-mFc was captured on a chip with covalentlyimmobilized antibody against murine IgG2a Fc and then exposed toAb-17G05 at concentrations ranging from 0.0195 μg/ml to 5 μg/ml. TheK_(D) was 2.8×10⁻¹¹ at 25° C. and 9.2×10⁻¹¹ at 37° C.

FIG. 3 depicts neutralizing activity of Ab-17G05 or Fab-17G05 in acell-based reporter gene assay. Included are assay responses withactivin A alone (5 ng/ml) and combined activin A and ActRIIB-Fc (50ng/ml). The potency of Ab-17G05 (IC₅₀≈0.04 nM) in neutralizing theinteraction between activin A and ActRIIB-Fc was approximately twoorders of magnitude higher than that of Fab-17G05 (IC₅₀≈2.6 nM).

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

Activins are dimeric polypeptide growth factors that belong to theTGF-beta superfamily. There are three principal activin forms (A, B, andAB) that are homo/heterodimers of two closely related β subunits(β_(A)β_(A), β_(B)β_(B), and β_(A)β_(B)). The human genome also encodesan activin C and an activin E, which are primarily expressed in theliver. In the TGF-beta superfamily, activins are unique andmultifunctional factors that can stimulate hormone production in ovarianand placental cells, support neuronal cell survival, influencecell-cycle progress positively or negatively depending on cell type, andinduce mesodermal differentiation at least in amphibian embryos (DePaoloet al., 1991, Proc Soc Ep Biol Med. 198:500-512; Dyson et al., 1997,Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol. 55:953-963). Inseveral tissues, activin signaling is antagonized by its relatedheterodimer, inhibin. For example, during the release offollicle-stimulating hormone (FSH) from the pituitary, activin promotesFSH secretion and synthesis, while inhibin prevents FSH secretion andsynthesis. Other proteins that may regulate activin bioactivity and/orbind to activin include follistatin (FS), follistatin-related protein(FSRP), α₂-macroglobulin, Cerberus, and endoglin. Together withmyostatin, activin has been implicated as a negative regulator ofskeletal muscle mass. He et al. 2005 Anat Embryol (Berl) 209:401-407;Link and Nishi, Exp. Cell Res. 1997 233:350-62.

TGF-β superfamily signals are mediated by heteromeric complexes of typeI and type II serine/threonine kinase receptors, which phosphorylate andactivate downstream Smad proteins upon ligand stimulation (Massague,2000, Nat. Rev. Mol. Cell Biol. 1:169-178). These type I and type IIreceptors are transmembrane proteins, composed of a ligand-bindingextracellular domain with cysteine-rich region, a transmembrane domain,and a cytoplasmic domain with predicted serine/threonine specificity.Type I receptors are essential for signaling; and type II receptors arerequired for binding ligands and for expression of type I receptors.Type I and II activin receptors form a stable complex after ligandbinding, resulting in phosphorylation of type I receptors by type IIreceptors.

Two related type II receptors, ActRIIA and ActRIIB, have been identifiedas the type II receptors for activins (Mathews and Vale, 1991, Cell65:973-982; Attisano et al., 1992, Cell 68: 97-108). Besides activins,ActRIIA and ActRIIB can interact biochemically with several other TGF-βfamily proteins, including BMP7, Nodal, BMP9, BMP10, GDF8, and GDF11(Yamashita et al., 1995, J. Cell Biol. 130:217-226; Lee and McPherron,2001, Proc. Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol.Cell 7: 949-957; Oh et al., 2002, Genes Dev. 16:2749-54). ALK4 is theprimary type I receptor for activins, particularly for activin A, andALK-7 may serve as a receptor for activins as well, particularly foractivin B.

Inhibitors of the activin signaling pathway have been proposed fortreatment of a variety of disorders, including bone loss, various tumorsincluding multiple myeloma and breast cancer, and anemia. Inhibitors ofmyostatin and GDF11 signaling have likewise been proposed for thetreatment of a variety of disorders, including muscle disorders,neurological disorders and bone disorders. Neutralizing anti-ActRIIBantibodies that interfere with signaling by any or all of activin A,activin B, GDF8 or GDF11 may be used in a variety of indications for thetreatment of muscle loss or insufficient muscle growth, includingmyopathies, muscular dystrophies, muscular atrophy, cachexia, andage-related conditions such as sarcopenia as well as for the treatmentof bone disorders and various cancers.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values. Alternatively, and particularly inbiological systems, the terms “about” and “approximately” may meanvalues that are within an order of magnitude, preferably within 5-foldand more preferably within 2-fold of a given value. Numerical quantitiesgiven herein are approximate unless stated otherwise, meaning that theterm “about” or “approximately” can be inferred when not expresslystated.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants(sequence variants). Such comparisons typically comprise alignments ofpolymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue. Unless indicated otherwise, BLAST shallbe the default algorithm for comparisons.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

As used herein, the term “ActRIIB” refers to a family of activinreceptor type IIB (ActRIIB) proteins from any species. Reference toActRIIB herein is understood to be a reference to any one of thecurrently identified forms. Members of the ActRIIB family are generallytransmembrane proteins, composed of a ligand-binding extracellulardomain with a cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine kinase activity.

The human ActRIIB precursor protein sequence is as follows (NCBIReference Sequence NM_001106.3), with the underlined sequencecorresponding to the literature-reported mature extracellular domain,within which are epitopes targeted by neutralizing anti-ActRIIBantibodies and other ActRIIB binding agents.

(SEQ ID NO: 1) MTAPWVALALLWGSLCAGSGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTLLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPPPSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTPGMKHENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLSYLHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQHPSLEELQEVVVHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAGCVEERVSLIRRSVNGTTSDCLVSLVTSV TNVDLPPKESSI

A protein comprising an extracellular domain of ActRIIB (ECD) maycomprise amino acids 19-134 of SEQ ID NO: 1, or smaller portions, suchas amino acids 20-134, 25-131 or any polypeptide comprising a portion ofSEQ ID NO: 1 beginning at any of amino acids 19-29 and ending at any ofamino acids 129-134. Each of the foregoing has been demonstrated toretain ligand binding activity. A protein comprising an extracellulardomain of ActRIIB may comprise a polypeptide that is at least 80, 85,90, 95, 96, 97, 98, 99 or 100% identical to any of the foregoing aminoacid sequence portions of SEQ ID NO: 1. An ActRIIB-Fc fusion protein isany protein comprising any of the foregoing extracellular domains ofActRIIB and an Fc portion of an immunoglobulin (e.g., IgG1, IgG2, IgG4),optionally including an interposed linker between the ActRIIB portionand the Fc portion.

The nucleic acid sequence encoding human ActRIIB precursor protein is asfollows (nucleotides 25-1560 of Genbank NM_001106.3):

(SEQ ID NO: 2) atgacggcgc cctgggtggc cctcgccctc ctctggggatcgctgtgcgc cggctctggg cgtggggagg ctgagacacgggagtgcatc tactacaacg ccaactggga gctggagcgcaccaaccaga gcggcctgga gcgctgcgaa ggcgagcaggacaagcggct gcactgctac gcctcctggc gcaacagctctggcaccatc gagctcgtga agaagggctg ctggctagatgacttcaact gctacgatag gcaggagtgt gtggccactgaggagaaccc ccaggtgtac ttctgctgct gtgaaggcaacttctgcaac gaacgcttca ctcatttgcc agaggctgggggcccggaag tcacgtacga gccacccccg acagcccccaccctgctcac ggtgctggcc tactcactgc tgcccatcgggggcctttcc ctcatcgtcc tgctggcctt ttggatgtaccggcatcgca agccccccta cggtcatgtg gacatccatgaggaccctgg gcctccacca ccatcccctc tggtgggcctgaagccactg cagctgctgg agatcaaggc tcgggggcgctttggctgtg tctggaaggc ccagctcatg aatgactttgtagctgtcaa gatcttccca ctccaggaca agcagtcgtggcagagtgaa cgggagatct tcagcacacc tggcatgaagcacgagaacc tgctacagtt cattgctgcc gagaagcgaggctccaacct cgaagtagag ctgtggctca tcacggccttccatgacaag ggctccctca cggattacct caaggggaacatcatcacat ggaacgaact gtgtcatgta gcagagacgatgtcacgagg cctctcatac ctgcatgagg atgtgccctggtgccgtggc gagggccaca agccgtctat tgcccacagggactttaaaa gtaagaatgt attgctgaag agcgacctcacagccgtgct ggctgacttt ggcttggctg ttcgatttgagccagggaaa cctccagggg acacccacgg acaggtaggcacgagacggt acatggctcc tgaggtgctc gagggagccatcaacttcca gagagatgcc ttcctgcgca ttgacatgtatgccatgggg ttggtgctgt gggagcttgt gtctcgctgcaaggctgcag acggacccgt ggatgagtac atgctgccctttgaggaaga gattggccag cacccttcgt tggaggagctgcaggaggtg gtggtgcaca agaagatgag gcccaccattaaagatcact ggttgaaaca cccgggcctg gcccagctttgtgtgaccat cgaggagtgc tgggaccatg atgcagaggctcgcttgtcc gcgggctgtg tggaggagcg ggtgtccctgattcggaggt cggtcaacgg cactacctcg gactgtctcgtttccctggt gacctctgtc accaatgtgg acctgccccc taaagagtca agcatc

2. ActRIIB Binding Agents

The disclosure provides binding agents (such as antibodies) thatspecifically bind to ActRIIB or portions of ActRIIB, and methods forusing such binding agents. The binding agents are useful to block orimpair the binding of human ActRIIB to one or more ligand(s) and tointerfere with its biological activity.

It will be understood by one of skill in the art that there is a highdegree of sequence identity between the orthologs of ActRIIB. Forexample, a murine ortholog of human ActRIIB has been described (NCBIRef. Seq.: NP_031423) that differs by only one amino acid substitutionin the mature ActRIIB extracellular domain (119 amino acids).Accordingly, agents binding to human ActRIIB will be expected to bind tomurine ActRIIB in cases where the recognition site of the binding agent,e.g., an antibody binding site such as an epitope, is highly conservedand in particular nearly or completely identical to the human sequence.Thus, when the term “specific binding to ActRIIB” is used, it isunderstood to include binding to multiple species of ActRIIB where thesequences between species are conserved.

Given the known structure of ActRIIB and the highly characterized ligandbinding interface (see, e.g., Weber et al. 2007, BMC Structural Biology7:6; Thompson et al. 2003 EMBO J. 22:1555-1566; WO 2006/012627), it isunderstood that neutralizing anti-ActRIIB antibodies will bind to aminoacids within one or more of the following strings of amino acids ofActRIIB as follows (numbering is relative to SEQ ID NO: 1): amino acids77-83 of SEQ ID NO: 1, amino acids 60-64 of SEQ ID NO: 1, 73-74 of SEQID NO: 1, amino acids 73-83 of SEQ ID NO: 1, amino acids 98-101 of SEQID NO: 1; amino acids 35-39 of SEQ ID NO:1 and/or amino acids 52-55 ofSEQ ID NO: 1.

Examples of binding agents according to the invention include theantibody 17G05 (Ab-17G05) and the corresponding Fab-17G05. As usedherein, Ab-17G05 comprises the polypeptides expressed by the nucleotidesshown in SEQ ID NOs: 17 and 18.

Binding agents of the invention are typically antibodies or fragmentsthereof, as defined herein. The term “antibody” refers to an intactantibody, or a binding fragment thereof. An antibody may comprise acomplete antibody molecule (including polyclonal, monoclonal, chimeric,humanized, or human versions having full-length heavy and/or lightchains), or comprise an antigen-binding fragment thereof. Antibodyfragments include F(ab′)₂, Fab, Fab′, Fv, Fc, and Fd fragments, and canbe incorporated into single-domain antibodies, single-chain antibodies,maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies,v-NAR and bis-scFv (See e.g., Hollinger and Hudson, 2005, NatureBiotechnology, 23, 9, 1 126-1136). Antibody-like polypeptides are alsodisclosed in U.S. Pat. No. 6,703,199 [“Artificial AntibodyPolypeptides”, assigned to Research Corp Technologies], includingfibronectin polypeptide monobodies. Other antibody-like polypeptides aredisclosed in U.S. patent publication 2005/0238646, which aresingle-chain polypeptides. As used herein, the isolated antibody or anantigen-binding fragment thereof may be a polyclonal antibody, amonoclonal antibody, a humanized antibody, a human antibody, a chimericantibody, or the like. In each of these types of binding agents, it isgenerally expected that one would insert one or more CDRs from theantibodies disclosed herein to produce an alternative ActRIIB bindingagent.

An antibody according to the present invention may belong to anyimmunoglobin class, for example IgG, IgE, IgM, IgD, or IgA. It may beobtained from or derived from an animal, for example, birds (e.g.,chicken) and mammals, which include but are not limited to a mouse, rat,hamster, rabbit, cow, horse, sheep, goat, camel, human, or otherprimate. The antibody may be an internalizing antibody. Within the humanIgG class, classes IgG1, IgG2 and IgG4 are particularly useful. Ananti-ActRIIB antibody of the IgG2 or IgG4 class may be particularlyuseful as a therapeutic as these classes will diminish the action of theimmune system against cells to which the anti-ActRIIB antibody binds.

Antigen binding fragments derived from an antibody can be obtained, forexample, by proteolytic hydrolysis of the antibody, for example, pepsinor papain digestion of whole antibodies according to conventionalmethods. By way of example, antibody fragments can be produced byenzymatic cleavage of antibodies with pepsin to provide a 5S fragmenttermed F(ab′)₂. This fragment can be further cleaved using a thiolreducing agent to produce 3.5S Fab monovalent fragments. Optionally, thecleavage reaction can be performed using a blocking group for thesulfhydryl groups that result from cleavage of disulfide linkages. As analternative, an enzymatic cleavage using papain produces two monovalentFab fragments and an Fc fragment directly. These methods are described,for example, by Goldenberg, U.S. Pat. No. 4,331,647, Nisonoff et al.,Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959;Edelman et al., in Methods in Enzymology 1:422 (Academic Press 1967);and by Andrews, S. M. and Titus, J. A. in Current Protocols inImmunology (Coligan J. E., et al., eds), John Wiley & Sons, New York(2003), pages 2.8.1-2.8.10 and 2.10A.1-2.10A.5. Other methods forcleaving antibodies, such as separating heavy chains to form monovalentlight-heavy chain fragments (Fd), further cleaving of fragments, orother enzymatic, chemical, or genetic techniques may also be used, solong as the fragments bind to the antigen that is recognized by theintact antibody.

An antibody fragment may also be any synthetic or genetically engineeredprotein. For example, antibody fragments include isolated fragmentsconsisting of the light chain variable region, “Fv” fragments consistingof the variable regions of the heavy and light chains, recombinantsingle-chain polypeptide molecules in which light and heavy variableregions are connected by a peptide linker (scFv proteins).

Another form of an antibody fragment is a peptide comprising one or morecomplementarity determining regions (CDRs) of an antibody. CDRs (alsotermed “minimal recognition units”, or “hypervariable regions”) can beobtained by constructing polynucleotides that encode the CDR ofinterest. Such polynucleotides are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region using mRNAof antibody-producing cells as a template (see, for example, Larrick etal., Methods: A Companion to Methods in Enzymology 2:106, 1991;Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies. Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995); andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Thus, in one embodiment, the binding agent comprises at least one CDR asdescribed herein. The binding agent may comprise at least two, three,four, five or six CDRs, as described herein. The binding agent furthermay comprise at least one variable region domain of an antibodydescribed herein. The variable region domain may be of any size or aminoacid composition and will generally comprise at least one CDR sequenceresponsible for binding to human ActRIIB, for example CDR-H1, CDR-H2,CDR-H3, and/or the light chain CDRs specifically described herein andwhich are adjacent to or in frame with one or more framework sequences.In general terms, the variable (V) region domain may be any suitablearrangement of immunoglobulin heavy (VH) and/or light (VL) chainvariable domains. Thus, for example, the V region domain may bemonomeric and be a VH or VL domain, which is capable of independentlybinding human ActRIIB with an affinity at least equal to 1×10⁻⁷ M orless as described below. Alternatively, the V region domain may bedimeric and contain VH-VH, VH-VL, or VL-VL dimers. The V region dimercomprises at least one VH and at least one VL chain that may benon-covalently associated (hereinafter referred to as FV). If desired,the chains may be covalently coupled either directly, for example via adisulfide bond between the two variable domains, or through a linker,for example a peptide linker, to form a single chain Fv (scFV).

The variable region domain may be any naturally occurring variabledomain or an engineered version thereof. By engineered version is meanta variable region domain that has been created using recombinant DNAengineering techniques. Such engineered versions include those created,for example, from a specific antibody variable region by insertions,deletions, or changes in or to the amino acid sequences of the specificantibody. Particular examples include engineered variable region domainscontaining at least one CDR and optionally one or more framework aminoacids from a first antibody and the remainder of the variable regiondomain from a second antibody.

The variable region domain may be covalently attached at a C-terminalamino acid to at least one other antibody domain or a fragment thereof.Thus, for example, a VH domain that is present in the variable regiondomain may be linked to an immunoglobulin CH1 domain, or a fragmentthereof. Similarly a VL domain may be linked to a CK domain or afragment thereof. In this way, for example, the antibody may be a Fabfragment wherein the antigen binding domain contains associated VH andVL domains covalently linked at their C-termini to a CH1 and CK domain,respectively. The CH1 domain may be extended with further amino acids,for example to provide a hinge region or a portion of a hinge regiondomain as found in a Fab fragment, or to provide further domains, suchas antibody CH2 and CH3 domains.

As described herein, binding agents may comprise at least one of theseCDRs. For example, one or more CDRs may be incorporated into knownantibody framework regions (IgG1, IgG2, etc.), or conjugated to asuitable vehicle to enhance the half-life thereof. Suitable vehiclesinclude, but are not limited to Fc, polyethylene glycol (PEG), albumin,transferrin, and the like. These and other suitable vehicles are knownin the art. Such conjugated CDR peptides may be in monomeric, dimeric,tetrameric, or other form. In one embodiment, one or more water-solublepolymer is bonded at one or more specific position, for example at theamino terminus, of a binding agent.

In certain embodiments, a binding agent comprises one or more watersoluble polymer attachments, including, but not limited to, polyethyleneglycol, polyoxyethylene glycol, or polypropylene glycol. See, e.g., U.S.Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and4,179,337. In certain embodiments, a derivative binding agent comprisesone or more of monomethoxy-polyethylene glycol, dextran, cellulose, orother carbohydrate-based polymers, poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol) and polyvinyl alcohol, as well as mixtures of suchpolymers. In certain embodiments, one or more water-soluble polymers israndomly attached to one or more side chains. In certain embodiments,PEG can act to improve the therapeutic capacity for a binding agent,such as an antibody. Certain such methods are discussed, for example, inU.S. Pat. No. 6,133,426, which is hereby incorporated by reference forany purpose.

Antibodies according to the invention may be obtained by conventionalimmunization and cell fusion procedures as described herein and known inthe art. Monoclonal antibodies of the invention may be generated using avariety of known techniques. In general, monoclonal antibodies that bindto specific antigens may be obtained by methods known to those skilledin the art (see, for example, Kohler et al., Nature 256:495, 1975;Coligan et al. (eds.), Current Protocols in Immunology, 1:2.5.12.6.7(John Wiley & Sons 1991); U.S. Pat. Nos. RE 32,011, 4,902,614,4,543,439, and 4,411,993; Monoclonal Antibodies, Hybridomas: A NewDimension in Biological Analyses, Plenum Press, Kennett, McKearn, andBechtol (eds.) (1980); and Antibodies: A Laboratory Manual, Harlow andLane (eds.), Cold Spring Harbor Laboratory Press (1988); Picksley etal., “Production of monoclonal antibodies against proteins expressed inE. coli,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover etal. (eds.), page 93 (Oxford University Press 1995)). Antibody fragmentsmay be derived therefrom using any suitable standard technique such asproteolytic digestion, or optionally, by proteolytic digestion (forexample, using papain or pepsin) followed by mild reduction of disulfidebonds and alkylation. Alternatively, such fragments may also begenerated by recombinant genetic engineering techniques as describedherein.

Monoclonal antibodies can be obtained by injecting an animal, forexample, a rat, hamster, rabbit, or mouse, with an immunogen comprisinghuman ActRIIB of SEQ ID NO: 1, or a fragment thereof, according tomethods known in the art and described herein. A polypeptide comprisingamino acids 19-134 or 20-134 of SEQ ID NO: 1 is particularly useful forgenerating antibodies that bind to the extracellular domain of ActRIIB,which includes the ligand binding portion. The presence of specificantibody production may be monitored after the initial injection and/orafter a booster injection by obtaining a serum sample and detecting thepresence of an antibody that binds to human ActRIIB or peptide using anyone of several immunodetection methods known in the art and describedherein. From animals producing the desired antibodies, lymphoid cells,most commonly cells from the spleen or lymph node, are removed to obtainB-lymphocytes. The B lymphocytes are then fused with a drug-sensitizedmyeloma cell fusion partner, preferably one that is syngeneic with theimmunized animal and that optionally has other desirable properties(e.g., inability to express endogenous Ig gene products, e.g., P3X63-Ag8.653 (ATCC No. CRL 1580); NSO, SP20) to produce hybridomas, which areimmortal eukaryotic cell lines. The lymphoid (e.g., spleen) cells andthe myeloma cells may be combined for a few minutes with a membranefusion-promoting agent, such as polyethylene glycol or a nonionicdetergent, and then plated at low density on a selective medium thatsupports the growth of hybridoma cells but not unfused myeloma cells. Apreferred selection media is HAT (hypoxanthine, aminopterin, thymidine).After a sufficient time, usually about one to two weeks, colonies ofcells are observed. Single colonies are isolated, and antibodiesproduced by the cells may be tested for binding activity to humanActRIIB, using any one of a variety of immunoassays known in the art anddescribed herein. The hybridomas are cloned (e.g., by limited dilutioncloning or by soft agar plaque isolation) and positive clones thatproduce an antibody specific to ActRIIB are selected and cultured. Themonoclonal antibodies from the hybridoma cultures may be isolated fromthe supernatants of hybridoma cultures. An alternative method forproduction of a murine monoclonal antibody is to inject the hybridomacells into the peritoneal cavity of a syngeneic mouse, for example, amouse that has been treated (e.g., pristane-primed) to promote formationof ascites fluid containing the monoclonal antibody. Monoclonalantibodies can be isolated and purified by a variety of well-establishedtechniques. Such isolation techniques include affinity chromatographywith protein-A Sepharose, size-exclusion chromatography, andion-exchange chromatography (see, for example, Coligan at pages2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., “Purification ofImmunoglobulin G (IgG),” in Methods in Molecular Biology, Vol. 10, pages79-104 (The Humana Press, Inc. 1992)). Monoclonal antibodies may bepurified by affinity chromatography using an appropriate ligand whoseselection is based on particular properties of the antibody (e.g.,heavy- or light-chain isotype, binding specificity, etc.). Examples of asuitable ligand, immobilized on a solid support, include Protein A,Protein G, an anti-constant region (light chain or heavy chain)antibody, an anti-idiotype antibody, or fragment or variant thereof.

It will be appreciated by one of skill in the art that a binding agentof the present invention may have at least one amino acid substitution,providing that the binding agent retains binding specificity. Therefore,modifications to the binding agent structures are encompassed within thescope of the invention. These may include amino acid substitutions,which may be conservative or non-conservative and that do not destroythe ActRIIB binding capability of a binding agent. Conservative aminoacid substitutions may encompass non-naturally occurring amino acidresidues, which are typically incorporated by chemical peptide synthesisrather than by synthesis in biological systems. These includepeptidomimetics and other reversed or inverted forms of amino acidmoieties. A conservative amino acid substitution may also involve asubstitution of a native amino acid residue with a normative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position.

Conservative substitutions are shown in Table 1 under the heading of“preferred substitutions”. If such substitutions result in a change inbiological activity, then more substantial changes, denominated“exemplary substitutions” in Table 1, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 1 Conservative Substitutions Original Preferred Residue ExemplarySubstitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln;Asn Lys Asn (N) Gln; His; Asp; Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C)Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala AlaHis (H) Arg; Asn; Gln; Lys Arg Ile (I) Leu; Val; Met; Ala; Phe;Norleucine Leu Leu (L) Ile; Norleucine; Val; Met; Ala; Phe Ile Lys (K)Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Tyr; Trp; Leu; Val;Ile; Ala Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser; Val Ser Trp(W) Tyr; Phe Tyr Tyr (Y) Phe; Trp; Thr; Ser Phe Val (V) Leu; Ile;Norleucine; Met; Phe; Ala Leu

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: Met, Ala, Val, Leu, Ile, Norleucine;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro; and

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions may involve the exchange of a member ofone class of amino acids or amino acid mimetics for a member fromanother class with different physical properties (e.g. size, polarity,hydrophobicity, charge). Such substituted residues may be introducedinto regions of the human antibody that are homologous with non-humanantibodies, or into the nonhomologous regions of the molecule.

Moreover, one skilled in the art may generate test variants containing asingle amino acid substitution at each desired amino acid residue. Thevariants can then be screened using activity assays as described herein.Such variants could be used to gather information about suitablevariants. For example, if one discovered that a change to a particularamino acid residue resulted in destroyed, undesirably reduced, orunsuitable activity, variants with such a change may be avoided. Inother words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

A skilled artisan will be able to determine suitable variants of thepolypeptide as set forth herein using well-known techniques. In certainembodiments, one skilled in the art may identify suitable areas of themolecule that may be changed without destroying activity by targetingregions not believed to be important for activity. In certainembodiments, one can identify residues and portions of the moleculesthat are conserved among similar polypeptides. In certain embodiments,even areas that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a protein that correspond toamino acid residues which are important for activity or structure insimilar proteins. One skilled in the art may opt for chemically similaramino acid substitutions for such predicted important amino acidresidues.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult J., Curr. Op. in Biotech.,7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974);Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv.Enzymol. Relat. Areas MoI. Biol., 47: 45-148 (1978); Chou et al., Ann.Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384(1979). Moreover, computer programs are currently available to assistwith predicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsequence similarity greater than 40% often have similar structuraltopologies. The recent growth of the protein structural database (PDB)has provided enhanced predictability of secondary structure, includingthe potential number of folds within a polypeptide's or protein'sstructure. See Holm et al., Nucl. Acid. Res., 27(1):244-247 (1999). Ithas been suggested (Brenner et al., Curr. Op. Struct. Biol.,7(3):369-376 (1997)) that there are a limited number of folds in a givenpolypeptide or protein and that once a critical number of structureshave been resolved, structural prediction will become dramatically moreaccurate.

Additional methods of predicting secondary structure include “threading”(Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al.,Structure, 4(1):15-19 (1996)), “profile analysis” (Bowie et al.,Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159(1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358(1987)), and “evolutionary linkage” (See Holm, supra (1999), andBrenner, supra (1997)).

It will be understood by one skilled in the art that some proteins, suchas antibodies, may undergo a variety of posttranslational modificationsduring expression and secretion from host cells. The type and extent ofthese modifications often depends on the host cell line used to expressthe protein as well as the culture conditions. Such modifications mayinclude variations in glycosylation, methionine or tryptophan oxidation,diketopiperizine formation, aspartate isomerization and asparaginedeamidation. A frequent modification is the loss of a carboxy-terminalbasic residue (such as lysine or arginine) due to the action ofcarboxypeptidases (as described in Harris, RJ. Journal of Chromatography705:129-134, 1995). Once the proteins have been expressed and processedthey are in a ‘mature’ form. Thus it is understood that the inventionincludes mature antibodies that result from expression of the DNAs ofthe invention.

In certain embodiments, variants of binding agents include glycosylationvariants wherein the number and/or type of glycosylation site has beenaltered compared to the amino acid sequences of a parent polypeptide. Incertain embodiments, variants comprise a greater or a lesser number ofN-linked glycosylation sites than the native protein. An N-linkedglycosylation site is characterized by the sequence: Asn-X-Ser orAsn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution of amino acidresidues to create this sequence provides a potential new site for theaddition of an N-linked carbohydrate chain. Alternatively, substitutionswhich eliminate this sequence will remove an existing N-linkedcarbohydrate chain. Also provided is a rearrangement of N-linkedcarbohydrate chains wherein one or more N-linked glycosylation sites(typically those that are naturally occurring) are eliminated and one ormore new N-linked sites are created. Additional preferred antibodyvariants include cysteine variants wherein one or more cysteine residuesare deleted from or substituted for another amino acid (e.g., serine) ascompared to the parent amino acid sequence. Cysteine variants may beuseful when antibodies must be refolded into a biologically activeconformation such as after the isolation of insoluble inclusion bodies.Cysteine variants generally have fewer cysteine residues than the nativeprotein, and typically have an even number to minimize interactionsresulting from unpaired cysteines.

Amino acid substitutions (whether conservative or non-conservative) canbe determined by those skilled in the art at the time such substitutionsare desired. In certain embodiments, amino acid substitutions can beused to identify important residues of antibodies to ActRIIB, or toincrease or decrease the affinity of the antibodies to ActRIIB describedherein.

According to certain embodiments, preferred amino acid substitutions arethose which: (1) reduce susceptibility to proteolysis, (2) reducesusceptibility to oxidation, (3) alter binding affinity for formingprotein complexes, (4) alter binding affinities, and/or (4) confer ormodify other physiochemical or functional properties on suchpolypeptides. According to certain embodiments, single or multiple aminoacid substitutions (in certain embodiments, conservative amino acidsubstitutions) may be made in the naturally-occurring sequence (incertain embodiments, in the portion of the polypeptide outside thedomain(s) forming intermolecular contacts). In certain embodiments, aconservative amino acid substitution typically may not substantiallychange the structural characteristics of the parent sequence (e.g., areplacement amino acid should not tend to break a helix that occurs inthe parent sequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W.H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al. Nature 354:105 (1991), which are each incorporatedherein by reference.

In certain embodiments, binding agents of the invention may bechemically bonded with polymers, lipids, or other moieties.

The binding agents may comprise at least one of the CDRs describedherein incorporated into a biocompatible framework structure. In oneexample, the biocompatible framework structure comprises a polypeptideor portion thereof that is sufficient to form a conformationally stablestructural support, or framework, or scaffold, which is able to displayone or more sequences of amino acids (e.g., CDRs, a variable region,etc.) that bind to an antigen in a localized surface region. Suchstructures can be a naturally occurring polypeptide or polypeptide“fold” (a structural motif), or can have one or more modifications, suchas additions, deletions or substitutions of amino acids, relative to anaturally occurring polypeptide or fold. These scaffolds can be derivedfrom a polypeptide of any species (or of more than one species), such asa human, other mammal, other vertebrate, invertebrate, plant, bacteria,or virus.

Typically the biocompatible framework structures are based on proteinscaffolds or skeletons other than immunoglobulin domains. For example,those based on fibronectin, ankyrin, lipocalin, neocarzinostain,cytochrome b, CPl zinc finger, PST1, coiled coil, LACl-Dl, Z domain andtendramisat domains may be used (See e.g., Nygren and Uhlen, 1997,Current Opinion in Structural Biology, 7, 463-469).

In preferred embodiments, it will be appreciated that the binding agentsof the invention include humanized antibodies, which can be producedusing techniques known to those skilled in the art (Zhang, W., et al.,Molecular Immunology. 42(12): 1445-1451, 2005; Hwang W. et al., Methods.36(1):35-42, 2005; Dall'Acqua W F, et al., Methods 36(1):43-60, 2005;and Clark, M., Immunology Today. 21(8):397-402, 2000).

An antibody of the present invention may also be a human monoclonalantibody. Human monoclonal antibodies may be generated by any number oftechniques with which those having ordinary skill in the art will befamiliar. Such methods include, but are not limited to, Epstein BarrVirus (EBV) transformation of human peripheral blood cells (e.g.,containing B lymphocytes), in vitro immunization of human B cells,fusion of spleen cells from immunized transgenic mice carrying insertedhuman immunoglobulin genes, isolation from human immunoglobulin V regionphage libraries, or other procedures as known in the art and based onthe disclosure herein. For example, human monoclonal antibodies may beobtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge. Methodsfor obtaining human antibodies from transgenic mice are described, forexample, by Green et al., Nature Genet. 7:13, 1994; Lonberg et al.,Nature 368:856, 1994; Taylor et al., Int. Immun. 6:579, 1994; U.S. Pat.No. 5,877,397; Bruggemann et al., 1997 Curr. Opin. Biotechnol. 8:455-58;Jakobovits et al., 1995 Ann. N. Y Acad. Sci. 764:525-35. In thistechnique, elements of the human heavy- and light-chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy-chain andlight-chain loci (see also Bruggemann et al., Curr. Opin. Biotechnol.8:455-58 (1997)). For example, human immunoglobulin transgenes may bemini-gene constructs, or transloci on yeast artificial chromosomes,which undergo B cell-specific DNA rearrangement and hypermutation in themouse lymphoid tissue. Human monoclonal antibodies may be obtained byimmunizing the transgenic mice, which may then produce human antibodiesspecific for ActRIIB. Lymphoid cells of the immunized transgenic micecan be used to produce human antibody-secreting hybridomas according tothe methods described herein. Polyclonal sera containing humanantibodies may also be obtained from the blood of the immunized animals.

Another method for generating human antibodies of the invention includesimmortalizing human peripheral blood cells by EBV transformation. See,e.g., U.S. Pat. No. 4,464,456. Such an immortalized B cell line (orlymphoblastoid cell line) producing a monoclonal antibody thatspecifically binds to ActRIIB can be identified by immunodetectionmethods as provided herein, for example, an ELISA, and then isolated bystandard cloning techniques. The stability of the lymphoblastoid cellline producing an anti-ActRIIB antibody may be improved by fusing thetransformed cell line with a murine myeloma to produce a mouse-humanhybrid cell line according to methods known in the art (see, e.g.,Glasky et al., Hybridoma 8:377-89 (1989)). Still another method togenerate human monoclonal antibodies is in vitro immunization, whichincludes priming human splenic B cells with human ActRIIB, followed byfusion of primed B cells with a heterohybrid fusion partner. See, e.g.,Boerner et al., 1991, J. Immunol. 147:86-95.

In certain embodiments, a B cell that is producing an anti-human ActRIIBantibody is selected and the light chain and heavy chain variableregions are cloned from the B cell according to molecular biologytechniques known in the art (WO 92/02551; U.S. Pat. No. 5,627,052;Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)) anddescribed herein. B cells from an immunized animal may be isolated fromthe spleen, lymph node, or peripheral blood sample by selecting a cellthat is producing an antibody that specifically binds to ActRIIB. Bcells may also be isolated from humans, for example, from a peripheralblood sample. Methods for detecting single B cells that are producing anantibody with the desired specificity are well known in the art, forexample, by plaque formation, fluorescence-activated cell sorting, invitro stimulation followed by detection of specific antibody, and thelike. Methods for selection of specific antibody-producing B cellsinclude, for example, preparing a single cell suspension of B cells insoft agar that contains human ActRIIB. Binding of the specific antibodyproduced by the B cell to the antigen results in the formation of acomplex, which may be visible as an immunoprecipitate. After the B cellsproducing the desired antibody are selected, the specific antibody genesmay be cloned by isolating and amplifying DNA or mRNA according tomethods known in the art and described herein.

Additionally, one skilled in the art will recognize that suitablebinding agents include portions of these antibodies, such as one or moreof CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, as specificallydisclosed herein. At least one of the regions of CDR-H1, CDR-H2, CDR-H3,CDR-L1, CDR-L2 and CDR-L3 may have at least one amino acid substitution,provided that the binding agent retains the binding specificity of thenon-substituted CDR. CDRs may be altered to increase or decrease lengthas well, and thus changes that are characterized as substitutions,insertions and deletions are all contemplated. The non-CDR portion ofthe binding agent may be a non-protein molecule, wherein the bindingagent cross-blocks the binding of an antibody disclosed herein toActRIIB and/or neutralizes ActRIIB. The non-CDR portion of the bindingagent may be composed of amino acids, wherein the binding agent is arecombinant binding protein or a synthetic peptide, and the recombinantbinding protein cross-blocks the binding of an antibody disclosed hereinto ActRIIB and/or neutralizes ActRIIB. The non-CDR portion of thebinding agent may be composed of amino acids, wherein the binding agentis a recombinant binding protein, and the recombinant binding proteinexhibits a similar binding pattern to human ActRIIB peptides in thehuman ActRIIB peptide epitope competition binding assay (describedhereinbelow) as that exhibited by antibody Ab-17G05, and/or neutralizesActRIIB.

In one embodiment, it is contemplated that one can use the antibodyheavy chain as ‘bait’ in a library screen where the library is composedof human antibody light chains, to identify complementing human lightchains where the reconstituted antibody binds to ActRIIB. In thisembodiment, the heavy chain is from an antibody specific to ActRIIB andis mouse, chimeric, or humanized.

Where an antibody comprises one or more of CDR-H1, CDR-H2, CDR-H3,CDR-L1, CDR-L2, and CDR-L3, as described above, it may be obtained byexpression from a host cell containing DNA coding for these sequences. ADNA coding for each CDR sequence may be determined on the basis of theamino acid sequence of the CDR and synthesized together with any desiredantibody variable region framework and constant region DNA sequencesusing oligonucleotide synthesis techniques, site-directed mutagenesisand polymerase chain reaction (PCR) techniques as appropriate. DNAcoding for variable region frameworks and constant regions is widelyavailable to those skilled in the art from genetic sequences databasessuch as GenBank®.

Once synthesized, the DNA encoding an antibody of the invention orfragment thereof may be propagated and expressed according to any of avariety of well-known procedures for nucleic acid excision, ligation,transformation, and transfection using any number of known expressionvectors. Thus, in certain embodiments expression of an antibody fragmentmay be preferred in a prokaryotic host, such as Escherichia coli (see,e.g., Pluckthun et al., 1989 Methods Enzymol. 178:497-515). In certainother embodiments, expression of the antibody or a fragment thereof maybe preferred in a eukaryotic host cell, including yeast (e.g.,Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichiapastoris), animal cells (including mammalian cells) or plant cells.Examples of suitable animal cells include, but are not limited to,myeloma (such as a mouse NSO line), COS, CHO, or hybridoma cells.Examples of plant cells include tobacco, corn, soybean, and rice cells.

One or more replicable expression vectors containing DNA encoding anantibody variable and/or constant region may be prepared and used totransform an appropriate cell line, for example, a non-producing myelomacell line, such as a mouse NSO line or a bacteria, such as E. coli, inwhich production of the antibody will occur. To obtain efficienttranscription and translation, the DNA sequence in each vector shouldinclude appropriate regulatory sequences, particularly a promoter andleader sequence operatively linked to the variable domain sequence.Particular methods for producing antibodies in this way are generallywell-known and routinely used. For example, basic molecular biologyprocedures are described by Maniatis et al. (Molecular Cloning, ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York,1989; see also Maniatis et al, 3rd ed., Cold Spring Harbor Laboratory,New York, (2001)). DNA sequencing can be performed as described inSanger et al. (PNAS 74:5463, (1977)) and the Amersham International picsequencing handbook, and site directed mutagenesis can be carried outaccording to methods known in the art (Kramer et al., Nucleic Acids Res.12:9441, (1984); Kunkel Proc. Natl. Acad. Sci. USA 82:488-92 (1985);Kunkel et al., Methods in Enzymol. 154:367-82 (1987); the AnglianBiotechnology Ltd handbook). Additionally, numerous publicationsdescribe techniques suitable for the preparation of antibodies bymanipulation of DNA, creation of expression vectors, and transformationand culture of appropriate cells (Mountain A and Adair, J R inBiotechnology and Genetic Engineering Reviews (ed. Tombs, M P, 10,Chapter 1, 1992, Intercept, Andover, UK); “Current Protocols inMolecular Biology”, 1999, F. M. Ausubel (ed.), Wiley Interscience, NewYork).

Where it is desired to improve the affinity of antibodies according tothe invention containing one or more of the above-mentioned CDRs,improved antibodies can be obtained by a number of affinity maturationprotocols including maintaining the CDRs (Yang et al., J. MoI. Biol.,254, 392-403, 1995), chain shuffling (Marks et al., Bio/Technology, 10,779-783, 1992), use of mutation strains of E. coli. (Low et al., J. MoI.Biol., 250, 350-368, 1996), DNA shuffling (Patten et al., Curr. Opin.Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. MoI.Biol., 256, 7-88, 1996) and sexual PCR (Crameri, et al., Nature, 391,288-291, 1998). All of these methods of affinity maturation arediscussed by Vaughan et al. (Nature Biotechnology, 16, 535-539, 1998).

An additional method for obtaining or maturing antibodies of theinvention is by phage display. See, e.g., Winter et al., 1994 Annu. Rev.Immunol. 12:433-55; Burton et al., 1994 Adv. Immunol. 57:191-280. Seealso the methodology described in the Examples. Combinatorial librariesof human or murine immunoglobulin variable-region genes may be createdin phage vectors that can be screened to select Ig fragments (Fab, Fv,sFv, or multimers thereof) that bind specifically to ActRIIB or variantor fragment thereof. See, e.g., U.S. Pat. No. 5,223,409; Huse et al.,1989 Science 246:1275-81; Sastry et al., Proc. Natl. Acad. Sci. USA86:5728-32 (1989); Alting-Mees et al., Strategies in Molecular Biology3:1-9 (1990); Kang et al., 1991 Proc. Natl. Acad. Sci. USA 88:4363-66;Hoogenboom et al., 1992 J. Molec. Biol. 227:381-388; Schlebusch et al.,1997 Hybridoma 16:47-52 and references cited therein; and Hoet et al.,2005, Nat Biotechnol 23:344-348. For example, a library containing aplurality of polynucleotide sequences encoding Ig variable regionfragments may be inserted into the genome of a filamentousbacteriophage, such as M 13 or a variant thereof, in frame with thesequence encoding a phage coat protein. A fusion protein may be a fusionof the coat protein with the light chain variable-region domain and/orwith the heavy chain variable-region domain. According to certainembodiments, immunoglobulin Fab fragments may also be displayed on aphage particle (see, e.g., U.S. Pat. No. 5,698,426).

Heavy and light chain immunoglobulin cDNA expression libraries may alsobe prepared in lambda phage, for example, using lambda ImmunoZap™ (H)and lambda ImmunoZap™ (L) vectors (Stratagene, La Jolla, Calif.).Briefly, mRNA is isolated from a B cell population, and used to createheavy and light chain immunoglobulin cDNA expression libraries in thelambda ImmunoZap(H) and lambda ImmunoZap(L) vectors. These vectors maybe screened individually or co-expressed to form Fab fragments orantibodies (see Huse et al., supra; see also Sastry et al., supra).Positive plaques may subsequently be converted to a non-lytic plasmidthat allows high level expression of monoclonal antibody fragments fromE. coli.

In one embodiment in a hybridoma, the variable regions of a geneexpressing a monoclonal antibody of interest are amplified usingnucleotide primers. These primers may be synthesized by one of ordinaryskill in the art, or may be purchased from commercially availablesources. (See, e.g., Stratagene (La Jolla, Calif.), which sells primersfor mouse and human variable regions including, among others, primersfor VHa, VHb, VHc, VHd, CHI, VL and CL regions.) These primers may beused to amplify heavy or light chain variable regions, which may then beinserted into vectors such as ImmunoZAP™ H or ImmunoZAP™ (Stratagene),respectively. These vectors may then be introduced into E. coli, yeast,or mammalian-based systems for expression. Large amounts of asingle-chain protein containing a fusion of the VH and VL domains may beproduced using these methods (see Bird et al., Science 242:423-426,1988).

Once cells producing antibodies according to the invention have beenobtained using any of the above-described immunization and othertechniques, the specific antibody genes may be cloned by isolating andamplifying DNA or mRNA therefrom according to standard procedures asdescribed herein. The antibodies produced therefrom may be sequenced andthe CDRs identified and the DNA coding for the CDRs may be manipulatedas described previously to generate other antibodies according to theinvention.

Preferably the binding agents bind specifically to ActRIIB. As with allbinding agents and binding assays, one of skill in this art recognizesthat the various moieties to which a binding agent should not detectablybind in order to be therapeutically effective and suitable would beexhaustive and impractical to list. Therefore, for a binding agentdisclosed herein, the term “specifically binds” refers to the ability ofa binding agent to bind to ActRIIB, preferably human ActRIIB, withgreater affinity than it binds to an unrelated control protein.Preferably the control protein is hen egg white lysozyme. Preferably thebinding agents bind to ActRIIB with an affinity that is at least, 50,100, 250, 500, 1000, or 10,000 times greater than the affinity for acontrol protein. A binding agent may have a binding affinity for humanActRIIB of less than or equal to 1×10⁻⁷ M, less than or equal to 1×10⁻⁸M, less than or equal to 1×10⁻⁹ M, less than or equal to 1×10⁻¹⁰ M, lessthan or equal to 1×10⁻¹¹ M, or less than or equal to 1×10⁻¹² M.Antibodies having improved affinity may be generated by any of a varietyof known maturation techniques, such as those described above. Affinitymay be assessed at different temperatures, using any of the techniquesdescribed herein. Temperatures of 20 deg. C., 25 deg. C. or 37 deg. C.may be used.

Affinity may be determined by an affinity ELISA assay. In certainembodiments, affinity may be determined by a BIACORE™ assay. In certainembodiments, affinity may be determined by a kinetic method. In certainembodiments, affinity may be determined by an equilibrium/solutionmethod. Such methods are described in further detail herein or known inthe art.

The affinity of a binding agent such as an antibody or binding partner,as well as the extent to which a binding agent (such as an antibody)inhibits binding, can be determined by one of ordinary skill in the artusing conventional techniques, for example by surface plasmon resonance(SPR; BIACORE™, Biosensor, Piscataway, N.J.) or according to methodsdescribed by Scatchard et al. (Ann. N.Y. Acad. Sci. 51:660-672 (1949)).For surface plasmon resonance, target molecules are immobilized on asolid phase and exposed to ligands in a mobile phase running along aflow cell. If ligand binding to the immobilized target occurs, the localrefractive index changes, leading to a change in SPR angle, which can bemonitored in real time by detecting changes in the intensity of thereflected light. The rates of change of the SPR signal can be analyzedto yield apparent rate constants for the association and dissociationphases of the binding reaction. The ratio of these values gives theapparent equilibrium constant (affinity) (see, e.g., Wolff et al.,Cancer Res. 53:2560-65 (1993)).

An oligopeptide or polypeptide is within the scope of the invention ifit comprises an amino acid sequence that is at least 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to at least one of theCDRs depicted in the Examples (SEQ ID NOs: 19-42); and/or to a CDR of anActRIIB binding agent that cross-blocks the binding of Ab-17G05 toActRIIB, and/or is cross-blocked from binding to ActRIIB by Ab-17G05;and/or to a CDR of an ActRIIB binding agent wherein the binding agentcan block the effect of ActRIIB in a cell-based assay (i.e. an ActRIIBneutralizing binding agent).

Examples of ActRIIB binding agent polypeptides and antibodies that arewithin the scope of the invention are those that have amino acidsequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to a variable region ofAb-17G05 (SEQ ID NOs: 15 and 16), and cross-block the binding ofAb-17G05 to ActRIIB, and/or are cross-blocked from binding to ActRIIB byAb-17G05; and/or can block the inhibitory effect of ActRIIB in acell-based assay (i.e. an ActRIIB neutralizing binding agent).

Examples of polynucleotides encoding ActRIIB binding agents that arewithin the scope of the invention are those that have polynucleotidesequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide encoding avariable region of Ab-17G05 (SEQ ID NOs: 17 and 18), and wherein theencoded ActRIIB binding agents cross-block the binding of Ab-17G05 toActRIIB, and/or are cross-blocked from binding to ActRIIB by Ab-17G05;and/or can block the inhibitory effect of ActRIIB in a cell-based assay(i.e. an ActRIIB neutralizing binding agent).

ActRIIB binding agents of the present invention preferably modulateActRIIB function in the cell-based assay described herein and/or the invivo assay described herein and/or cross-block the binding of antibodyAb-17G05 described in this application and/or are cross-blocked frombinding ActRIIB by the antibody Ab-17G05 described in this application.Accordingly, such binding agents can be identified using the assaysdescribed herein.

In certain embodiments, binding agents are generated by firstidentifying antibodies that neutralize ActRIIB in the cell-based and/orin vivo assays described herein and/or cross-block the antibody Ab-17G05described in this application and/or are cross-blocked from bindingActRIIB by antibody Ab-17G05 described in this application. The CDRregions from these antibodies are then used to insert into appropriatebiocompatible frameworks to generate ActRIIB binding agents. The non-CDRportion of the binding agent may be composed of amino acids, or may be anonprotein molecule. The assays described herein allow thecharacterization of binding agents. Preferably the binding agents of thepresent invention are antibodies as defined herein.

In the methods described herein to generate antibodies according to theinvention, including the manipulation of the specific Ab-17G05 CDRs intonew frameworks and/or constant regions, appropriate assays are availableto select the desired antibodies or binding agents (i.e. assays fordetermining binding affinity to ActRIIB; cross-blocking assays such asthe BIACORE™-based human ActRIIB peptide competition binding assaysdescribed in Example 2 below; A204 cell-based assay; in vivo assays).

The terms “cross-block”, “cross-blocked” and “cross-blocking” are usedinterchangeably herein to mean the ability of an antibody or otherbinding agent to interfere with the binding of other antibodies orbinding agents to ActRIIB.

The extent to which an antibody or other binding agent is able tointerfere with the binding of another to ActRIIB, and therefore whetherit can be said to cross-block according to the invention, can bedetermined using competition binding assays. One particularly suitablequantitative assay uses a BIACORE™ instrument which can measure theextent of interactions using surface plasmon resonance technology.Example 2 provides methods for conducting a BIACORE™ basedcross-blocking assays. Another suitable quantitative cross-blockingassay uses an ELISA-based approach to measure competition betweenantibodies or other binding agents in terms of their binding to ActRIIB.

The following generally describes a suitable BIACORE™ assay fordetermining whether an antibody or other binding agent cross-blocks oris capable of cross-blocking according to the invention. Forconvenience, reference is made to two antibodies, but it will beappreciated that the assay can be used with any of the ActRIIB bindingagents described herein. The BIACORE™ instrument (for example theBIACORE™ 3000) is operated according to the manufacturer'srecommendations.

Thus, in one cross-blocking assay, ActRIIB-mFc fusion protein iscaptured on a CM5 BIACORE™ chip by previously attached anti-mFc IgG togenerate an ActRIIB-coated surface. Typically 200-800 resonance units ofActRIIB-mFc (dimeric) would be coupled to the chip (an amount that giveseasily measurable levels of binding but that is readily saturable by theconcentrations of test reagent being used).

The two antibodies (termed A* and B*) to be assessed for their abilityto cross-block each other are mixed at a one to one molar ratio ofbinding sites in a suitable buffer to create the test mixture. Whencalculating the concentrations on a binding site basis the molecularweight of an antibody is assumed to be the total molecular weight of theantibody divided by the number of ActRIIB binding sites on thatantibody.

The concentration of each antibody in the test mix should be high enoughto readily saturate the binding sites for that antibody on theActRIIB-mFc molecules captured on the BIACORE™ chip. The antibodies inthe mixture are at the same molar concentration (on a binding basis) andthat concentration would typically be between 1.00 and 1.5 micromolar(on a binding site basis).

Separate solutions containing antibody A* alone and antibody B* aloneare also prepared. Antibody A* and antibody B* in these solutions shouldbe in the same buffer and at the same concentration as in the test mix.

The test mixture is passed over the ActRIIB-mFc-coated BIACORE™ chip andthe total amount of binding recorded. The chip is then treated in such away as to remove the bound antibodies without damaging the chip-boundActRIIB-mFc. Typically, this is done by treating the chip with 30 mM HClfor 60 seconds.

The solution of antibody A* alone is then passed over theActRIIB-mFc-coated surface and the amount of binding recorded. The chipis again treated to remove all of the bound antibody without damagingthe chip-bound ActRIIB-mFc.

The solution of antibody B* alone is then passed over theActRIIB-mFc-coated surface and the amount of binding recorded.

The maximum theoretical binding of the mixture of antibody A* andantibody B* is next calculated, and is the sum of the binding of eachantibody when passed over the ActRIIB surface alone. If the actualrecorded binding of the mixture is less than this theoretical maximumthen the two antibodies are cross-blocking each other.

Thus, in general, a cross-blocking antibody or other binding agentaccording to the invention is one which will bind to ActRIIB in theabove BIACORE™ cross-blocking assay such that during the assay and inthe presence of a second antibody or other binding agent of theinvention the recorded binding is between 80% and 0.1% (e.g. 80% to 4%)of the maximum theoretical binding, specifically between 75% and 0.1%(e.g. 75% to 4%) of the maximum theoretical binding, and morespecifically between 70% and 0.1% (e.g. 70% to 4%) of maximumtheoretical binding (as just defined above) of the two antibodies orbinding agents in combination.

The BIACORE™ assay described above is an assay used to determine ifantibodies or other binding agents cross-block each other according tothe invention. On rare occasions, particular antibodies or other bindingagents may not bind to ActRIIB-mFc coupled via anti-mFc IgG to a CM5BIACORE™ chip (this might occur when the relevant binding site onActRIIB is masked or destroyed by ActRIIB linkage to mFc). In suchcases, cross-blocking can be determined using a tagged version ofActRIIB, for example C-terminal His-tagged ActRIIB. In this particularformat, an anti-His antibody would be coupled to the BIACORE™ chip andthen the His-tagged ActRIIB would be passed over the surface of the chipand captured by the anti-His antibody. The cross-blocking analysis wouldbe carried out essentially as described above, except that after eachchip regeneration cycle, new His-tagged ActRIIB would be loaded backonto the surface coated with anti-His antibody. Moreover, various othertags and tag binding protein combinations that are known in the artcould be used for such a cross-blocking analysis (e.g. HA tag withanti-HA antibodies; FLAG tag with anti-FLAG antibodies; biotin tag withstreptavidin).

The following generally describes an ELISA assay for determining whetheran anti-ActRIIB antibody or other ActRIIB binding agent cross-blocks oris capable of cross-blocking according to the invention. Forconvenience, reference is made to two antibodies, but it will beappreciated that the assay can be used with any of the ActRIIB bindingagents described herein.

The general principle of the assay is to have an anti-ActRIIB antibodycoated onto the wells of an ELISA plate. An excess amount of a second,potentially cross-blocking, anti-ActRIIB antibody is added in solution(i.e. not bound to the ELISA plate). A limited amount of ActRIIB (oralternatively ActRIIB-mFc) is then added to the wells. The coatedantibody and the antibody in solution compete for binding of the limitednumber of ActRIIB (or ActRIIB-mFc) molecules. The plate is washed toremove ActRIIB that has not been bound by the coated antibody and toalso remove the second, solution-phase antibody as well as any complexesformed between the second, solution-phase antibody and ActRIIB. Theamount of bound ActRIIB is then measured using an appropriate ActRIIBdetection reagent. An antibody in solution that is able to cross-blockthe coated antibody will be able to cause a decrease in the number ofActRIIB molecules that the coated antibody can bind relative to thenumber of ActRIIB molecules that the coated antibody can bind in theabsence of the second, solution-phase antibody.

This assay is described here in more detail for Ab-17G05 and atheoretical antibody Ab-XX. In the instance where Ab-17G05 is chosen tobe the immobilized antibody, it is coated onto the wells of the ELISAplate, after which the plates are blocked with a suitable blockingsolution to minimize non-specific binding of reagents that aresubsequently added. An excess amount of Ab-XX is then added to the ELISAplate such that the moles of Ab-XX ActRIIB binding sites per well are atleast 10-fold higher than the moles of Ab-17G05 ActRIIB binding sitesthat were used, per well, during the coating of the ELISA plate.

ActRIIB is then added such that the moles of ActRIIB added per well areat least 25-fold lower than the moles of Ab-17G05 ActRIIB binding sitesthat were used for coating each well. Following a suitable incubationperiod the ELISA plate is washed and an ActRIIB detection reagent isadded to measure the amount of ActRIIB specifically bound by the coatedanti-ActRIIB antibody (in this case Ab-17G05). The background signal forthe assay is defined as the signal obtained in wells with the coatedantibody (in this case Ab-17G05), solution-phase antibody (in this caseAb-XX), ActRIIB buffer only (i.e. no ActRIIB) and ActRIIB detectionreagents. The positive control signal for the assay is defined as thesignal obtained in wells with the coated antibody (in this caseAb-17G05), solution-phase antibody buffer only (i.e. no solution-phaseantibody), ActRIIB and ActRIIB detection reagents. The ELISA assay needsto be run in such a manner so as to have the positive control signal atleast 3 times the background signal.

As a control for methodologic artifacts, the cross-blocking assay may berun in the format just described and also reversed, with Ab-XX as thecoated antibody and Ab-17G05 as the solution-phase antibody.

A reporter gene assay in A204 cells may be used to determine the abilityof anti-ActRIIB Fabs and recombinant antibodies to neutralize ActRIIB.This assay is based on a human rhabdomyosarcoma cell line transfectedwith a pGL3(CAGA)12 reporter plasmid (Dennler et al, 1998, EMBO 17:3091-3100) as well as a Renilla reporter plasmid (pRLCMV) to control fortransfection efficiency. The CAGA12 motif is present in TGF-betaresponsive genes (PAI-1 gene), so this vector is of general use forfactors signaling through Smad2 and Smad3. Since the A204 cell lineexpresses primarily ActRIIA rather than ActRIIB, it is not possible todirectly test antibodies for potential ActRIIB neutralizing ability.Instead, this assay is designed to detect the ability of test articlesto neutralize the inhibitory effect of the soluble fusion proteinActRIIB-Fc on activation of endogenous ActRIIA by ligands (such asactivin A, myostatin or GDF11) that can bind with high affinity to bothActRIIA and ActRIIB. Thus, in this assay, ligand-mediated activation ofActRIIA will occur despite the presence of ActRIIB-Fc if theanti-ActRIIB Fab or antibody is neutralizing.

On the first day of the assay, A204 cells (ATCC HTB-82) are distributedin 48-well plates at 10⁵ cells per well. On the second day, a solutioncontaining 10 μg pGL3(CAGA)12, 1 g pRLCMV, 30 μl Fugene 6 (RocheDiagnostics), and 970 μl OptiMEM (Invitrogen) is preincubated for 30min, then added to McCoy's growth medium, which is applied to the platedcells (500 μl/well) for incubation overnight at room temperature. On thethird day, medium is removed, and cells are incubated for 6 h at 37° C.with a mixture of ligands and inhibitors prepared as described below.

To evaluate the neutralizing potency of Fabs or recombinant antibodies,a serial dilution of the test article is made in a 48-well plate in a200 μl volume of assay buffer (McCoy's medium+0.1% BSA). An equal volumeof ActRIIB-Fc (200 μg/ml) in assay buffer is then added. The testsolutions are incubated at 37° C. for 30 minutes, then 400 μl of GDF11(10 ng/ml) or activin A (10 ng/ml) is added to all wells, and 350 μl ofthis mixture is added to each well of the 48-well plate of A204 cells.Each concentration of Fab or antibody is tested in duplicate. The finalconcentration of ActRIIB-Fc is 50 ng/ml (which is the IC50 for thisinhibitor of activin A signaling when the final concentration of activinA is 5 ng/ml). After incubation with test solutions for 6 h, cells arerinsed with phosphate-buffered saline containing 0.1% BSA, then lysedwith passive lysis buffer (Promega E1941) and stored overnight at −70°C. On the fourth and final day, plates are warmed to room temperaturewith gentle shaking. Cell lysates are transferred in duplicate to achemoluminescence plate (96-well) and analyzed in a luminometer withreagents from a Dual-Luciferase Reporter Assay system (Promega E1980) todetermine normalized luciferase activity.

Antibodies disclosed herein bind to regions of human ActRIIB which areimportant for the in vivo activity of the protein, thereby inhibitingthe activity of ActRIIB. Ab-17G05 binds to an epitope within thesequence of amino acids 20-134 of SEQ ID NO: 1. Binding of an antibodyto ActRIIB can be correlated with changes in biomarkers associated withActRIIB-mediated signaling, for example, serum FSH levels, bone density,muscle dimensions (or mass or strength), or body weight.

Pharmacodynamic parameters dependent on ActRIIB signaling can bemeasured as endpoints for in vivo testing of ActRIIB binding agents inorder to identify those binding agents that are able to neutralizeActRIIB and provide a therapeutic benefit. An ActRIIB neutralizingbinding agent is defined as one capable of causing a statisticallysignificant change, as compared to vehicle-treated animals, in such apharmacodynamic parameter. Such in vivo testing can be performed in anysuitable mammal (e.g. mouse, rat, monkey).

3. Screening Assays and Other Biochemical Uses

In certain aspects, the present invention relates to the use of thesubject ActRIIB binding agents to identify compounds (agents) which areagonist or antagonists of ActRIIB. Compounds identified through thisscreening can be tested to assess their ability to modulateActRIIB-mediated signaling in vivo or in vitro. These compounds can betested, for example, in animal models.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. In aspecific embodiment, the test agent is a small organic molecule having amolecular weight of less than about 2,000 Daltons.

The test compounds of the invention can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase (GST),photoactivatible crosslinkers or any combinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between an ActRIIBbinding agent and an ActRIIB polypeptide.

Merely to illustrate, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with an isolated andpurified ActRIIB binding agent which is ordinarily capable of binding toan ActRIIB polypeptide, as appropriate for the intention of the assay.To the mixture of the compound and ActRIIB binding agent is then added acomposition containing an ActRIIB polypeptide. Detection andquantification of complexes between ActRIIB polypeptide and ActRIIBbinding agent provides a means for determining the compound's efficacyat inhibiting (or potentiating) complex formation between the ActRIIBpolypeptide and ActRIIB binding agent. The efficacy of the compound canbe assessed by generating dose response curves from data obtained usingvarious concentrations of the test compound. Moreover, a control assaycan also be performed to provide a baseline for comparison. For example,in a control assay, isolated and purified ActRIIB binding agent is addedto a composition containing an ActRIIB polypeptide, and the formation ofcomplexes between ActRIIB polypeptide and ActRIIB binding agent isquantitated in the absence of the test compound. It will be understoodthat, in general, the order in which the reactants may be admixed can bevaried, and can be admixed simultaneously. Moreover, in place ofpurified proteins, cellular extracts and lysates may be used to render asuitable cell-free assay system.

Complex formation between ActRIIB polypeptide and ActRIIB binding agentmay be detected by a variety of techniques. For instance, modulation ofthe formation of complexes can be quantitated using, for example,detectably labeled proteins such as radiolabeled (e.g., ³²P, ³⁵S, ¹⁴C or³H), fluorescently labeled (e.g., FITC), or enzymatically labeledActRIIB polypeptide or ActRIIB binding agent, by immunoassay, or bychromatographic detection.

4. Formulation and Delivery of Therapeutics

Pharmaceutical compositions are provided, comprising one of theabove-described binding agents such as antibody Ab-17G05 or a humanizedversion thereof, along with a pharmaceutically or physiologicallyacceptable carrier, excipient, or diluent.

The development of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., subcutaneous, oral, parenteral, intravenous,intranasal, and intramuscular administration and formulation, is wellknown in the art, some of which are briefly discussed below for generalpurposes of illustration.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein subcutaneously,parenterally, intravenously, intramuscularly, or even intraperitoneally.Such approaches are well known to the skilled artisan, some of which arefurther described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat.No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain embodiments,solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations generally will contain a preservative to prevent the growthof microorganisms.

Illustrative pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants. Prevention ofthe action of microorganisms can be facilitated by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, Remington's PharmaceuticalSciences, 15th ed., pp. 1035-1038 and 1570-1580). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. Moreover, for human administration, preparations will ofcourse preferably meet sterility, pyrogenicity, and the general safetyand purity standards as required by FDA Office of Biologies standards.

In another embodiment of the invention, the compositions disclosedherein may be formulated in a neutral or salt form. Illustrativepharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective.

The carriers can further comprise any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The phrase “pharmaceutically-acceptable” refersto molecular entities and compositions that do not produce an allergicor similar untoward reaction when administered to a human.

In certain embodiments, liposomes, nanocapsules, microparticles, lipidparticles, vesicles, and the like, are used for the introduction of thecompositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

The formation and use of liposome and liposome-like preparations aspotential drug carriers is generally known to those of skill in the art(see for example, Lasic, Trends Biotechnol. 16(7):307-21, 1998;Takakura, Nippon Rinsho 56(3):691-95, 1998; Chandran et al., Indian J.Exp. Biol. 35(8):801-09, 1997; Margalit, Crit. Rev. Ther. Drug CarrierSyst. 12(2-3):233-61, 1995; U.S. Pat. No. 5,567,434; U.S. Pat. No.5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S.Pat. No. 5,795,587, each specifically incorporated herein by referencein its entirety). The use of liposomes does not appear to be associatedwith autoimmune responses or unacceptable toxicity after systemicdelivery. In certain embodiments, liposomes are formed fromphospholipids that are dispersed in an aqueous medium and spontaneouslyform multilamellar concentric bilayer vesicles (also termedmultilamellar vesicles (MLVs)).

Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev. Ind. Pharm. 24(12): 1113-28, 1998). To avoid side effectsdue to intracellular polymeric overloading, such ultrafine particles(sized around 0.1 um) may be designed using polymers able to be degradedin vivo. Such particles can be made as described, for example, byCouvreur et al., Crit. Rev. Ther. Drug Carrier Syst. 5(1):1-20, 1988;zur Muhlen et al., Eur. J. Pharm. Biopharm. 45(2):149-55, 1998; Zambauxet al., J Controlled Release 50(1-3):31-40, 1998; and U.S. Pat. No.5,145,684.

In addition, pharmaceutical compositions of the present invention may beplaced within containers, along with packaging material that providesinstructions regarding the use of such pharmaceutical compositions.Generally, such instructions will include a tangible expressiondescribing the reagent concentration, as well as within certainembodiments, relative amounts of excipient ingredients or diluents(e.g., water, saline or PBS) that may be necessary to reconstitute thepharmaceutical composition.

The dose administered may range from 0.01 mg/kg to 200 mg/kg of bodyweight, and optionally between 0.5 mg/kg and 20 mg/kg. However, as willbe evident to one of skill in the art, the amount and frequency ofadministration will depend, of course, on such factors as the nature andseverity of the indication being treated, the desired response, thecondition of the patient, and so forth. Typically, the compositions maybe administered by a variety of techniques, as noted above.

5. Therapeutic Uses of ActRIIB Binding Agents

In certain embodiments, ActRIIB binding agents of the present inventioncan be used for treating or preventing a disease or condition that isassociated with abnormal activity of ActRIIB and/or an ActRIIB ligand(e.g., activin A, GDF8, or GDF11). These diseases, disorders orconditions are generally referred to herein as “ActRIIB-associatedconditions.” In certain embodiments, the present invention providesmethods of treating or preventing a disease, disorder, or condition inan individual in need thereof through administering to the individual atherapeutically effective amount of an ActRIIB binding agent asdescribed above. These methods are particularly aimed at therapeutic andprophylactic treatments of animals, and more particularly, humans.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes prophylaxis of the named condition or amelioration orelimination of the condition once it has been established.

ActRIIB and ActRIIB-ligand complexes play essential roles in tissuegrowth as well as early developmental processes such as the correctformation of various structures or in one or more post-developmentalcapacities including sexual development, pituitary hormone production,and creation of bone and cartilage. Thus, ActRIIB-associated conditionsinclude abnormal tissue growth and developmental defects. In addition,ActRIIB-associated conditions include, but are not limited to, disordersof cell growth and differentiation such as inflammation, allergy,autoimmune diseases, infectious diseases, and tumors.

Exemplary ActRIIB-associated conditions include neuromuscular disorders(e.g., muscular dystrophy and muscle atrophy), congestive obstructivepulmonary disease or pulmonary emphysema (and associated musclewasting), muscle wasting syndrome, sarcopenia, cachexia, adipose tissuedisorders (e.g., obesity), type 2 diabetes, and bone degenerativedisease (e.g., osteoporosis). Other exemplary ActRIIB-associatedconditions include musculodegenerative and neuromuscular disorders,tissue repair (e.g., wound healing), neurodegenerative diseases (e.g.,amyotrophic lateral sclerosis), immunologic disorders (e.g., disordersrelated to abnormal proliferation or function of lymphocytes), andobesity or disorders related to abnormal proliferation of adipocytes.

In certain embodiments, ActRIIB binding agents of the invention are usedas part of a treatment for a muscular dystrophy. The term “musculardystrophy” refers to a group of degenerative muscle diseasescharacterized by gradual weakening and deterioration of skeletal musclesand sometimes the heart and respiratory muscles. Muscular dystrophiesare genetic disorders characterized by progressive muscle wasting andweakness that begin with microscopic changes in the muscle. As musclesdegenerate over time, the person's muscle strength declines.

Exemplary muscular dystrophies that can be treated with a regimenincluding the subject ActRIIB binding agents include: Duchenne musculardystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifussmuscular dystrophy (EDMD), limb-girdle muscular dystrophy (LGMD),fascioscapulohumeral muscular dystrophy (FSH or FSHD) (also known asLandouzy-Dejerine), myotonic muscular dystrophy (MMD) (also known asSteinert's Disease), oculopharyngeal muscular dystrophy (OPMD), distalmuscular dystrophy (DD), congenital muscular dystrophy (CMD), andscapulohumeral muscular dystrophy (SMD).

Duchenne muscular dystrophy (DMD) was first described by the Frenchneurologist Guillaume Benjamin Amand Duchenne in the 1860s. Beckermuscular dystrophy (BMD) is named after the German doctor Peter EmilBecker, who first described this variant of DMD in the 1950s. DMD is oneof the most frequent inherited diseases in males, affecting one in 3,500boys. DMD occurs when the dystrophin gene, located on the short arm ofthe X chromosome, is broken. Since males only carry one copy of the Xchromosome, they only have one copy of the dystrophin gene. Without thedystrophin protein, muscle is easily damaged during cycles ofcontraction and relaxation. While early in the disease musclecompensates by regeneration, later on muscle progenitor cells cannotkeep up with the ongoing damage and healthy muscle is replaced bynon-functional fibro-fatty tissue.

BMD results from different mutations in the dystrophin gene. BMDpatients have some dystrophin, but it is either insufficient in quantityor poor in quality. Having some dystrophin protects the muscles of thosewith BMD from degenerating as badly or as quickly as those of peoplewith DMD.

For example, studies demonstrate that blocking or eliminating functionof GDF8 (an ActRIIB ligand) in vivo can effectively treat at leastcertain symptoms in DMD and BMD patients. Thus, the subject ActRIIBbinding agents may act as GDF8 inhibitors (antagonists), and constitutean alternative means of blocking the functions of GDF8 and/or ActRIIB invivo in DMD and BMD patients.

In other embodiments, ActRIIB binding agents may also be used to treator prevent muscular atrophy due to myopathies, examples of which includeinflammatory myopathy, metabolic myopathy, and myotonia. Subject ActRIIBbinding agents have application in treating congenital myopathies suchas myotubular myopathy, nemalene myopathy, and mitochondrial myopathy.The subject ActRIIB binding agents may be used to treat inclusion bodymyositis, myoglobinurias, rhabdomyolysis, myositis ossificans,polymyositis, or dermatomyositis. In addition, ActRIIB binding agentsmay treat or prevent muscle atrophy arising from glucocorticoidtreatment, sarcopenia, prolonged bed rest, skeletal immobilization,sepsis, or congestive heart failure.

The subject ActRIIB binding agents provide an effective means toincrease muscle mass in other neuromuscular diseases or conditions thatare in need of muscle growth. For example, amyotrophic lateral sclerosis(ALS, also known as Lou Gehrig's disease or motor neuron disease) is achronic, incurable, and unstoppable CNS disorder that attacks the motorneurons, components of the CNS that connect the brain to the skeletalmuscles. In ALS, the motor neurons deteriorate and eventually die, andthough a person's brain normally remains fully functioning and alert,the command to move cannot reach the muscles. Most people who developALS are between 40 and 70 years old. The first motor neurons that weakenare those leading to the arms or legs. Those with ALS may have troublewalking, they may drop things, fall, slur their speech, and laugh or cryuncontrollably. Eventually the muscles in the limbs begin to atrophyfrom disuse. This muscle weakness will become debilitating and a personwill need a wheel chair or become unable to function out of bed. MostALS patients die from respiratory failure or from complications ofventilator assistance like pneumonia, 3-5 years from disease onset.Other neuromuscular diseases in which ActRIIB binding agents may beuseful include paralysis due to spinal cord injury or stroke;denervation due to trauma or degenerative, metabolic, or inflammatoryneuropathy; adult motor neuron disease; autoimmune motor neuropathy withmultifocal conductor block; and infantile or juvenile spinal muscularatrophy.

Increased muscle mass induced by ActRIIB binding agents might alsobenefit those suffering from muscle wasting diseases. Gonzalez-Cadavidet al. (1998, Proc. Natl. Acad. Sci. USA 95:14938-43) reported that thatGDF8 expression correlates inversely with fat-free mass in humans andthat increased expression of the GDF8 gene is associated with weightloss in men with AIDS wasting syndrome. By inhibiting the function ofGDF8 in AIDS patients, at least certain symptoms of AIDS may bealleviated, if not completely eliminated, thus significantly improvingquality of life in AIDS patients.

The cancer anorexia-cachexia syndrome is among the most debilitating andlife-threatening aspects of cancer. Progressive weight loss in canceranorexia-cachexia syndrome is a common feature of many types of cancerand is responsible not only for a poor quality of life and poor responseto chemotherapy, but also a shorter survival time than is found inpatients with comparable tumors without weight loss. Associated withanorexia, fat and muscle tissue wasting, psychological distress, and alower quality of life, cachexia arises from a complex interactionbetween the cancer and the host. It is one of the most common causes ofdeath among cancer patients and is present in 80% at death. It is acomplex example of metabolic chaos effecting protein, carbohydrate, andfat metabolism. Tumors produce both direct and indirect abnormalities,resulting in anorexia and weight loss. Currently, there is no treatmentto control or reverse the process. Cancer anorexia-cachexia syndromeaffects cytokine production, release of lipid-mobilizing andproteolysis-inducing factors, and alterations in intermediarymetabolism. Although anorexia is common, a decreased food intake aloneis unable to account for the changes in body composition seen in cancerpatients, and increasing nutrient intake is unable to reverse thewasting syndrome. Cachexia is generally suspected in patients withcancer if an involuntary weight loss of greater than five percent ofpremorbid weight occurs within a six-month period.

Since systemic overexpression of GDF8 in adult mice was found to induceprofound muscle and fat loss analogous to that seen in human cachexiasyndromes (Zimmers et al., 2002, Science 296:1486-1488), the subjectActRIIB binding agents as pharmaceutical compositions can bebeneficially used to prevent, treat, or alleviate the symptoms of thecachexia syndrome, where muscle growth is desired. This would includecachexia associated with cancer as well as cachexia associated withrheumatoid arthritis.

In other embodiments, the present invention provides methods of inducingbone and/or cartilage formation, preventing bone loss, increasing bonemineralization or preventing the demineralization of bone. For example,the subject ActRIIB binding agents identified in the present inventionhave application in treating osteoporosis and the healing of bonefractures and cartilage defects in humans and other animals. ActRIIBbinding agents may be useful in patients that are diagnosed withsubclinical low bone density, as a protective measure against thedevelopment of osteoporosis.

In one specific embodiment, methods and compositions of the presentinvention may find medical utility in the healing of bone fractures andcartilage defects in humans and other animals. The subject methods andcompositions may also have prophylactic use in closed as well as openfracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma-induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery. Further, methods and compositions of the invention maybe used in the treatment of periodontal disease, and in other toothrepair processes. In certain cases, the subject ActRIIB binding agentsmay provide an environment to attract bone-forming cells, stimulategrowth of bone-forming cells or induce differentiation of progenitors ofbone-forming cells. ActRIIB binding agents of the invention may also beuseful in the treatment of osteoporosis. Further, ActRIIB binding agentsmay be used in cartilage defect repair and prevention/reversal ofosteoarthritis.

In another specific embodiment, the invention provides a therapeuticmethod and composition for repairing fractures and other conditionsrelated to cartilage and/or bone defects or periodontal diseases. Theinvention further provides therapeutic methods and compositions forwound healing and tissue repair. The types of wounds include, but arenot limited to, burns, incisions and ulcers. See e.g., PCT PublicationNo. WO84/01106. Such compositions comprise a therapeutically effectiveamount of at least one of the ActRIIB binding agents of the invention inadmixture with a pharmaceutically acceptable vehicle, carrier or matrix.

In another specific embodiment, methods and compositions of theinvention can be applied to conditions causing bone loss such asosteoporosis, hyperparathyroidism, Cushing's disease, thyrotoxicosis,chronic diarrheal state or malabsorption, renal tubular acidosis, oranorexia nervosa. Many people know that being female, having a low bodyweight, and leading a sedentary lifestyle are risk factors forosteoporosis (loss of bone mineral density, leading to fracture risk).However, osteoporosis can also result from the long-term use of certainmedications. Osteoporosis resulting from drugs or another medicalcondition is known as secondary osteoporosis. In a condition known asCushing's disease, the excess amount of cortisol produced by the bodyresults in osteoporosis and fractures. The most common medicationsassociated with secondary osteoporosis are the corticosteroids, a classof drugs that act like cortisol, a hormone produced naturally by theadrenal glands. Although adequate levels of thyroid hormones (which areproduced by the thyroid gland) are needed for the development of theskeleton, excess thyroid hormone can decrease bone mass over time.Antacids that contain aluminum can lead to bone loss when taken in highdoses by people with kidney problems, particularly those undergoingdialysis. Other medications that can cause secondary osteoporosisinclude phenytoin (Dilantin) and barbiturates that are used to preventseizures; methotrexate (Rheumatrex, Immunex, Folex PFS), a drug for someforms of arthritis, cancer, and immune disorders; cyclosporine(Sandimmune, Neoral), a drug used to treat some autoimmune diseases andto suppress the immune system in organ transplant patients; luteinizinghormone-releasing hormone agonists (Lupron, Zoladex), used to treatprostate cancer and endometriosis; heparin (Calciparine, Liquaemin), ananticlotting medication; and cholestyramine (Questran) and colestipol(Colestid), used to treat high cholesterol. Gum disease causes bone lossbecause these harmful bacteria in our mouths force our bodies to defendagainst them. The bacteria produce toxins and enzymes under thegum-line, causing a chronic infection.

In a further embodiment, the present invention provides methods andtherapeutic agents for treating diseases or disorders associated withabnormal or unwanted bone growth. For example, patients having thedisease known as Fibrodysplasia Ossificans Progressiva (FOP) grow anabnormal “second skeleton” that prevents any movement. Additionally,abnormal bone growth can occur after hip replacement surgery and thusruin the surgical outcome. This is a more common example of pathologicalbone growth and a situation in which the subject methods andcompositions may be therapeutically useful. The same methods andcompositions may also be useful for treating other forms of abnormalbone growth (e.g., pathological growth of bone following trauma, burnsor spinal cord injury), and for treating or preventing the undesirableconditions associated with the abnormal bone growth seen in connectionwith metastatic prostate cancer or osteosarcoma. Examples of thesetherapeutic agents include, but are not limited to, ActRIIB bindingagents that specifically bind to an ActRIIB receptor such that anActRIIB ligand cannot bind to the ActRIIB receptor.

In other embodiments, the present invention provides compositions andmethods for regulating body fat content in an animal and for treating orpreventing conditions related thereto, and particularly,health-compromising conditions related thereto. According to the presentinvention, to regulate (control) body weight can refer to reducing orincreasing body weight, reducing or increasing the rate of weight gain,or increasing or reducing the rate of weight loss, and also includesactively maintaining, or not significantly changing body weight (e.g.,against external or internal influences which may otherwise increase ordecrease body weight). One embodiment of the present invention relatesto regulating body weight by administering to an animal (e.g., a human)in need thereof an ActRIIB binding agent.

In one specific embodiment, the present invention relates to methods andActRIIB binding agents for reducing body weight and/or reducing weightgain in an animal, and more particularly, for treating or amelioratingobesity in patients at risk for or suffering from obesity. Loss of GDF8(an ActRIIB ligand) function is associated with fat loss withoutdiminution of nutrient intake (McPherron et al., 1997, Proc. Natl. Acad.Sci. USA, 94:12457-12461). In another specific embodiment, the presentinvention is directed to methods and compounds for treating an animalthat is unable to gain or retain weight (e.g., an animal with a wastingsyndrome). Such methods are effective to increase body weight and/ormass, or to reduce weight and/or mass loss, or to improve conditionsassociated with or caused by undesirably low (e.g., unhealthy) bodyweight and/or mass. The subject ActRIIB binding agents may further beused as a therapeutic agent for slowing or preventing the development oftype II diabetes and metabolic syndrome.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments of thepresent invention, and are not intended to limit the invention.

Example 1 Selection, Prescreening, and Sequencing of ActRIIB-BindingFabs

A multi-round selection procedure was used to screen for Fabs thatdisplace ligand binding from human ActRIIB with high affinity. Dyax'sFab310 phage-display library (Hoet et al., 2005, Nat Biotechnol23:344-348) was screened with both a biotinylated human ActRIIB ECDtarget and a fusion protein target consisting of human ActRIIB ECDlinked to biotinylated human IgG1 Fc (ActRIIB-Fc), each targetpreviously immobilized on magnetic streptavidin beads and validated bySDS-polyacrylamide gel electrophoresis. To increase the probability ofobtaining ligand-blocking Fabs, an additional selection strategyinvolved multiple rounds in which the Fab library was ‘depleted’ byexposure to a complex consisting of biotinylated activin A bound tohistidine-tagged ActRIIB ECD. In this way, Fabs which bound to regionsoutside the ActRIIB ligand-binding site were preferentially removed fromthe library. Three rounds of such library depletion alternated withthree rounds of positive selection using either of the aforementionedtargets.

Individual selected clones were tested in a prescreening phage ELISA. Inthis assay, amplified phage supernatant from each clone was added toELISA plates onto which biotinylated ActRIIB had been immobilized, andbound M13 phage was detected with a horseradish-peroxidase-conjugatedantibody against the P8 major coat protein. Selection outputs yieldingpositive data from this prescreen ELISA analysis, defined as signalgreater than three times background, were carried forward forreformatting to generate soluble sFab-producing clones. Reformattinginvolves excision of gIII-encoding DNA from isolated phagemid vector toconvert Fab cassette DNA to a vector format suitable for sFab expressionin E. coli. Specifically, polyclonal phagemid DNA was isolated from eachselection output; gIII DNA was removed from circular, double-strandedphagemid DNA by restriction digestion with MluI; the linearized DNA waspurified and religated; and host E. coli was transformed with ligationproduct to obtain clonal transformants containing vector expressingsFab.

To identify clones expressing sFabs that bind ActRIIB, the reformattedclones were subjected to high-throughput plating, picking, andadditional screening by ELISA. This ELISA screening format differed fromthat described above for the Fab-phage ELISA in that supernatant fromsFab cultures was added to the ELISA wells and sFab bound to immobilizedActRIIB was detected by anti-Fab antibody. Those reformatted clonesdisplaying a sFab ELISA signal greater than two times background wererearrayed and subjected to confirmatory ELISA and high-throughput DNAsequencing of the VH and VL regions. Ninety-five sFab clones wereinitially identified as most promising leads based on the sFab ELISAanalysis and sequencing results described above.

Example 2 Characterization and Production of Lead ActRIIB-Binding Fabs

The 95 sFab clones were cultured on a small scale, affinity-purifiedwith protein A, and subjected to additional characterization. At Dyax,purified sFabs were immobilized on a surface plasmon resonance (SPR)microarray chip and exposed to ActRIIB ECD or ActRIIB-Fc fusion proteinin a high-throughput assay to determine approximate on- and off-rates.sFabs were ranked by off-rate (k_(d)) rather than equilibriumdissociation constant (K_(D)) due to bivalency of the target protein(confounding avidity effects), and thirteen sFabs were obtained withoff-rates less than 10⁻⁴ s⁻¹, as well as an additional 50 sFabs withoff-rates less than 10⁻³ s⁻¹. A competition assay was also performed toidentify sFabs that compete for the ligand-binding site on ActRIIB.Specifically, immobilized sFabs were exposed to a complex of ActRIIB ECDor ActRIIB-Fc fusion protein with activin A (in a ratio of 1 μM:100 nM)and the signal associated with sFab binding to this complex was comparedwith that of sFab binding to ActRIIB alone. sFabs whose signal wasreduced at least 50% by the presence of activin A were classified ascompetitors, and 72 of 95 sFabs met this criterion. Potentialcross-reactivity with ActRIIA-Fc was also evaluated by SPR and was foundto occur for 11 of 95 sFabs.

Affinity-purified sFabs were also screened in two SPR-based competitionassays at Acceleron. The first assay evaluated the effect of sFabpretreatment on binding of GDF11 to immobilized ActRIIB-Fc.Specifically, biotinylated ActRIIB-Fc was immobilized on a BIACORE™streptavidin chip and exposed to GDF11 (500 ng/ml) to determine the SPRsignal associated with maximum activin binding (Emax). This signal wascompared with a second signal (residual GDF11 binding) resulting fromGDF11 binding to immobilized ActRIIB-Fc that had first been exposed tosFab (40 μg/ml). In this assay, a Fab was considered neutralizing ifresidual GDF11 binding was less than 50% of Emax, and five sFabs (17A07,17A11, 17C09, 17G01, and 17G05) of the 95 screened met this criterion. Asecond assay evaluated the effect of sFab pretreatment on ActRIIBbinding to immobilized activin A. In this case, biotinylated activin Awas immobilized on a BIACORE™ streptavidin chip and exposed toActRIIB-Fc (1 g/ml) to determine the SPR signal associated with maximumactivin A binding (Emax). This signal was compared with residual bindingof activin A to a complex of sFab with ActRIIB-Fc, which was formed bypremixing these proteins in a 20:1 ratio (20 μg/ml Fab and 1 μg/mlActRIIB-Fc). sFabs were ranked according to residual binding, and 19 of95 sFabs screened exhibited residual binding less than 50% of Emax inthis assay.

Based on the foregoing analyses, 24 sFab clones with preferredcharacteristics (such as slow off-rate, inhibition of ligand binding,and low cross-reactivity with ActRIIA) were chosen for scaled-upproduction. Protein levels of approximately 50 μg were obtained for themajority of clones after one-step purification with protein A. PurifiedsFab proteins were subjected to confirmatory characterization by ELISAand SDS-polyacrylamide gel electrophoresis before transfer to Acceleronin these larger quantities. DNA sequences of the 24 preferred cloneswere also confirmed at this stage by standard methods.

Example 3 Sequences of Lead ActRIIB-Binding Fabs

Of the 24 sFabs with preferred characteristics, four were selected formore detailed characterization. Shown below are the amino acid sequencesof VH and VL, respectively, for Fab-17A11 (CDR sequences areunderlined).

(SEQ ID NO: 3)   1EVQLLESGGG LVQPGGSLRL SCAASGFTFS TYAMMWVRQA PGKGLEWVSR  51IYPSGGTTTY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGS 101AASSYWGQGT LVTVSS (SEQ ID NO: 4)   1QDIQMTQSPS FLSASVGDRV TITCRASQGI SNYLAWYQQK PGKAPKLLIY  51AASTLQSGVP SRFSGSGSGT EFTLTISSLQ PEDIGTYYCQ QLISYPFTFG 101 PGTKVDIKShown below are nucleotide sequences encoding VH and VL, respectively,for Fab-17A11.

(SEQ ID NO: 5)   1GAAGTTCAAT TGTTAGAGTC TGGTGGCGGT CTTGTTCAGC CTGGTGGTTC  51TTTACGTCTT TCTTGCGCTG CTTCCGGATT CACTTTCTCT ACTTACGCTA 101TGATGTGGGT TCGCCAAGCT CCTGGTAAAG GTTTGGAGTG GGTTTCTCGT 151ATCTATCCTT CTGGTGGCAC TACTACTTAT GCTGACTCCG TTAAAGGTCG 201CTTCACTATC TCTAGAGACA ACTCTAAGAA TACTCTCTAC TTGCAGATGA 251ACAGCTTAAG GGCTGAGGAC ACGGCCGTGT ATTACTGTGC GAGGGGATCA 301GCTGCCAGCT CCTACTGGGG CCAGGGAACC CTGGTCACCG TCTCAAGC (SEQ ID NO: 6)   1CAAGACATCC AGATGACCCA GTCTCCATCC TTCCTGTCTG CATCTGTTGG  51AGACAGGGTC ACCATCACTT GCCGGGCCAG TCAGGGCATT AGCAATTATT 101TAGCCTGGTA TCAGCAAAAA CCAGGGAAAG CCCCTAAGCT CCTGATCTAT 151GCTGCATCCA CTTTGCAAAG TGGGGTCCCA TCAAGGTTCA GCGGCAGTGG 201ATCTGGGACA GAATTCACTC TCACAATCAG CAGCCTGCAG CCTGAAGATA 251TTGGAACTTA TTACTGTCAA CAGCTTATTA GTTACCCATT CACTTTCGGC 301CCTGGGACCA AAGTGGATAT CAAShown below are the amino acid sequences of VH and VL, respectively, forFab-17C09 (CDR sequences are underlined).

(SEQ ID NO: 7)   1EVQLLESGGG LVQPGGSLRL SCAASGFTFS QYNMTWVRQA PGKGLEWVSS  51IYSSGGVTPY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGR 101LLFDYWGQGT LVTVSS (SEQ ID NO: 8)   1QDIQMTQSPS SLSASVGDRV TITCRASQSI SNYLNWYQQR PGKPPKLLIY  51AASSLQSGVP SRFSGSGSGT DFSLSISILQ PEDFATYYCQ QGYTAPRSFG 101 QGTKVEIKShown below are nucleotide sequences encoding VH and VL, respectively,for Fab-17C09.

(SEQ ID NO: 9)   1GAAGTTCAAT TGTTAGAGTC TGGTGGCGGT CTTGTTCAGC CTGGTGGTTC  51TTTACGTCTT TCTTGCGCTG CTTCCGGATT CACTTTCTCT CAGTACAATA 101TGACTTGGGT TCGCCAAGCT CCTGGTAAAG GTTTGGAGTG GGTTTCTTCT 151ATCTATTCTT CTGGTGGCGT TACTCCTTAT GCTGACTCCG TTAAAGGTCG 201CTTCACTATC TCTAGAGACA ACTCTAAGAA TACTCTCTAC TTGCAGATGA 251ACAGCTTAAG GGCTGAGGAC ACGGCCGTGT ATTACTGTGC GAGAGGTCGC 301CTCCTCTTTG ACTACTGGGG CCAGGGAACC CTGGTCACCG TCTCAAGC (SEQ ID NO: 10)   1CAAGACATCC AGATGACCCA GTCTCCATCC TCCCTGTCTG CATCTGTCGG  51AGACAGAGTC ACCATCACTT GCCGGGCAAG TCAGAGCATT AGCAACTATT 101TAAATTGGTA TCAGCAGAGA CCAGGGAAAC CCCCTAAGCT CCTGATCTAT 151GCTGCATCCA GTTTGCAAAG TGGGGTCCCA TCAAGGTTTA GCGGCAGTGG 201ATCTGGGACA GATTTCAGTC TCTCCATCAG CATTCTGCAA CCTGAAGATT 251TTGCAACTTA CTACTGTCAA CAGGGTTACA CTGCCCCTCG CAGTTTTGGC 301CAGGGGACCA AGGTGGAGAT CAAShown below are the amino acid sequences of VH and VL, respectively, forFab-17G01 (CDR sequences are underlined).

(SEQ ID NO: 11)   1EVQLLESGGG LVQPGGSLRL SCAASGFTFS NYQMDWVRQA PGKGLEWVSY  51IGPSGGRTKY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCARGL 101YSFDYWGQGT LVTVSS (SEQ ID NO: 12)   1QDIQMTQSPS SLSASVGDRV TITCRAGQSI SNFLNWYQHT PGTGPKVLIY  51AASSLQSGVP SRFSGSGSGT EFTLTITNLQ PEDFATYYCQ QSYSTPFTFG 101 PGTKVDIKShown below are nucleotide sequences encoding VH and VL, respectively,for Fab-17G01.

(SEQ ID NO: 13)   1GAAGTTCAAT TGTTAGAGTC TGGTGGCGGT CTTGTTCAGC CTGGTGGTTC  51TTTACGTCTT TCTTGCGCTG CTTCCGGATT CACTTTCTCT AATTACCAGA 101TGGATTGGGT TCGCCAAGCT CCTGGTAAAG GTTTGGAGTG GGTTTCTTAT 151ATCGGTCCTT CTGGTGGCCG TACTAAGTAT GCTGACTCCG TTAAAGGTCG 201CTTCACTATC TCTAGAGACA ACTCTAAGAA TACTCTCTAC TTGCAGATGA 251ACAGCTTAAG GGCTGAGGAC ACAGCCACAT ATTACTGTGC GAGAGGATTG 301TACTCGTTTG ACTACTGGGG CCAGGGAACC CTGGTCACCG TCTCAAGC (SEQ ID NO: 14)   1CAAGACATCC AGATGACCCA GTCTCCATCC TCCCTGTCTG CATCTGTAGG  51AGACAGAGTC ACCATCACTT GCCGGGCAGG TCAGAGCATT AGCAACTTTT 101TAAATTGGTA TCAGCATACA CCAGGGACAG GCCCTAAAGT CCTGATCTAT 151GCTGCATCCA GTTTGCAAAG TGGGGTCCCA TCACGGTTCA GTGGCAGTGG 201ATCTGGGACA GAATTCACTC TCACCATCAC CAATCTGCAA CCTGAAGATT 251TTGCAACTTA CTACTGTCAA CAGAGTTACA GTACCCCATT CACTTTCGGC 301CCTGGGACCA AAGTGGATAT CAGShown below are the amino acid sequences of VH and VL, respectively, forFab-17G05 (CDR sequences are underlined).

(SEQ ID NO: 15)   1EVQLLESGGG LVQPGGSLRL SCAASGFTFS NYWMGWVRQA PGKGLEWVSY  51IRSSGGLTHY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCAKGL 101YSFDYWGQGT LVTVSS (SEQ ID NO: 16)   1QDIQMTQSPS SLSASVGDRV TITCRASQGV NNFLAWYQQK PGKAPRLLIY  51AASTLQSGVP SRFSGSGSGT DFTLSISNLQ PEDFATYYCQ QSYSTPRGFG 101 QGTKVEIKShown below are nucleotide sequences encoding VH and VL, respectively,for Fab-17G05.

(SEQ ID NO: 17)   1GAAGTTCAAT TGTTAGAGTC TGGTGGCGGT CTTGTTCAGC CTGGTGGTTC  51TTTACGTCTT TCTTGCGCTG CTTCCGGATT CACTTTCTCT AATTACTGGA 101TGGGTTGGGT TCGCCAAGCT CCTGGTAAAG GTTTGGAGTG GGTTTCTTAT 151ATCCGTTCTT CTGGTGGCCT TACTCATTAT GCTGACTCCG TTAAAGGTCG 201CTTCACTATC TCTAGAGACA ACTCTAAGAA TACTCTCTAC TTGCAGATGA 251ACAGCTTAAG GGCTGAGGAC ACAGCCACAT ATTACTGTGC GAAAGGACTA 301TATTCCTTTG ACTACTGGGG CCAGGGAACC CTGGTCACCG TCTCAAGC (SEQ ID NO: 18)   1CAAGACATCC AGATGACCCA GTCTCCATCT TCCCTGTCTG CTTCTGTAGG  51AGACAGAGTC ACCATCACTT GCCGGGCCAG TCAGGGCGTT AACAATTTTT 101TAGCCTGGTA TCAGCAAAAA CCAGGGAAGG CCCCTAGGCT CCTGATCTAT 151GCTGCATCCA CTTTGCAGAG TGGGGTCCCA TCAAGGTTCA GCGGCAGTGG 201ATCTGGGACA GATTTCACTC TCTCCATCAG CAACCTGCAG CCTGAAGACT 251TTGCAACTTA TTACTGTCAA CAGAGTTACA GTACCCCTCG GGGGTTCGGC 301CAAGGGACCA AGGTGGAAAT CAAListed below are CDR sequences for Fab-17A11.

CDR-H1 (SEQ ID NO: 19) TYAMM CDR-H2 (SEQ ID NO: 20) RIYPSGGTTTYADSVKGCDR-H3 (SEQ ID NO: 21) GSAASSY CDR-L1 (SEQ ID NO: 22) RASQGISNYLA CDR-L2(SEQ ID NO: 23) AASTLQS CDR-L3 (SEQ ID NO: 24) QQLISYPFTListed below are CDR sequences for Fab-17C09.

CDR-H1 (SEQ ID NO: 25) QYNMT CDR-H2 (SEQ ID NO: 26) SIYSSGGVTPYADSVKGCDR-H3 (SEQ ID NO: 27) GRLLFDY CDR-L1 (SEQ ID NO: 28) RASQSISNYLN CDR-L2(SEQ ID NO: 29) AASSLQS CDR-L3 (SEQ ID NO: 30) QQGYTAPRSListed below are CDR sequences for Fab-17G01.

CDR-H1 (SEQ ID NO: 31) NYQMD CDR-H2 (SEQ ID NO: 32) YIGPSGGRTKYADSVKGCDR-H3 (SEQ ID NO: 33) GLYSFDY CDR-L1 (SEQ ID NO: 34) RAGQSISNFLN CDR-L2(SEQ ID NO: 35) AASSLQS CDR-L3 (SEQ ID NO: 36) QQSYSTPFTListed below are CDR sequences for Fab-17G05.

CDR-H1 (SEQ ID NO: 37) NYWMG CDR-H2 (SEQ ID NO: 38) YIRSSGGLTHYADSVKGCDR-H3 (SEQ ID NO: 39) GLYSFDY CDR-L1 (SEQ ID NO: 40) RASQGVNNFLA CDR-L2(SEQ ID NO: 41) AASTLQS CDR-L3 (SEQ ID NO: 42) QQSYSTPRG

Example 4 Characterization of Lead Fab Binding to ActRIIB

Applicants used SPR (BIACORE™-based analysis) to more fully characterizebinding of lead sFabs to ActRIIB. FIG. 1 shows kinetic characterizationof Fab-17G05 binding to human ActRIIB-hFc (dimeric protein), and thebinding parameters for several lead sFabs are indicated below.

25° C. 37° C. Fab k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) k_(a) (M⁻¹s⁻¹)k_(d) (s⁻¹) K_(D) (M) 17G05  16 × 10⁵ 8.7 × 10⁻⁴ 5.5 × 10⁻¹⁰  26 × 10⁵3.1 × 10⁻³ 1.9 × 10⁻⁹ 575A-M31-E07 8.7 × 10⁵ 1.6 × 10⁻⁴ 18 × 10⁻¹⁰ 11 ×10⁵ 5.3 × 10⁻³   5 × 10⁻⁹ 17G01 4.6 × 10⁵  18 × 10⁻⁴ 38 × 10⁻¹⁰ 5.8 ×10⁵   12 × 10⁻³  21 × 10⁻⁹ 17A11  12 × 10⁵ 1.9 × 10⁻⁴ 16 × 10⁻¹⁰ 14 ×10⁵  14 × 10⁻³ 9.9 × 10⁻⁹Among the lead sFabs analyzed, Fab-17G05 displayed the best kineticparameters for off-rate and K_(D) at 37° C., and for K_(D) at 25° C.

Example 5 Reporter Gene Assay in A204 Cells

A reporter gene assay in A204 cells was used to determine the ability ofanti-ActRIIB Fabs and recombinant antibodies to neutralize ActRIIB. Thisassay is based on a human rhabdomyosarcoma cell line transfected with apGL3(CAGA)12 reporter plasmid (Dennler et al, 1998, EMBO 17: 3091-3100)as well as a Renilla reporter plasmid (pRLCMV) to control fortransfection efficiency. The CAGA12 motif is present in TGF-betaresponsive genes (PAI-1 gene), so this vector is of general use forfactors signaling through Smad2 and Smad3. Since the A204 cell lineexpresses primarily ActRIIA rather than ActRIIB, it is not possible todirectly test antibodies for potential ActRIIB neutralizing ability.Instead, this assay was designed to detect the ability of test articlesto neutralize the inhibitory effect of the soluble fusion proteinActRIIB-Fc on activation of endogenous ActRIIA by ligands (such asactivin A, GDF11 or myostatin) that can bind with high affinity to bothActRIIA and ActRIIB. Thus, in this assay, ligand-mediated activation ofActRIIA will occur despite the presence of ActRIIB-Fc if theanti-ActRIIB Fab or antibody is neutralizing.

On the first day of the assay, A204 cells (ATCC HTB-82) were distributedin 48-well plates at 10⁵ cells per well. On the second day, a solutioncontaining 10 μg pGL3(CAGA)12, 1 g pRLCMV, 30 μl Fugene 6 (RocheDiagnostics), and 970 μl OptiMEM (Invitrogen) was preincubated for 30min, then added to McCoy's growth medium, which was applied to theplated cells (500 μl/well) for incubation overnight at room temperature.On the third day, medium was removed, and cells were incubated for 6 hat 37° C. with a mixture of ligands and inhibitors prepared as describedbelow.

To evaluate the neutralizing potency of Fabs or recombinant antibodies,a serial dilution of the test article was made in a 48-well plate in a200 μl volume of assay buffer (McCoy's medium+0.1% BSA). An equal volumeof ActRIIB-Fc (200 μg/ml) in assay buffer was then added. The testsolutions were incubated at 37° C. for 30 minutes, then 400 μl of GDF11(10 ng/ml) or activin A (10 ng/ml) was added to all wells, and 350 μl ofthis mixture was added to each well of the 48-well plate of A204 cells.Each concentration of Fab or antibody was tested in duplicate. The finalconcentration of ActRIIB-Fc was 50 ng/ml (which is the IC50 for thisinhibitor of activin A signaling when the final concentration of activinA is 5 ng/ml). After incubation with test solutions for 6 h, cells wererinsed with phosphate-buffered saline containing 0.1% BSA, then lysedwith passive lysis buffer (Promega E1941) and stored overnight at −70°C. On the fourth and final day, plates were warmed to room temperaturewith gentle shaking. Cell lysates were transferred in duplicate to achemoluminescence plate (96-well) and analyzed in a luminometer withreagents from a Dual-Luciferase Reporter Assay system (Promega E1980) todetermine normalized luciferase activity.

This reporter gene assay was used to screen several of the lead sFabs.In two different assays with GDF11 as ligand, Fab-17G05 was a morepotent neutralizer of cellular signaling than were other sFabs tested,including Fab-17A11 and Fab-17G01.

Example 6 Generation of Ab-17G05 by Fab Conversion

On the basis of the foregoing results, Fab-17G05 was selected forconversion to an antibody. Construction of vectors for expression ofhuman IgG heavy and light chains was based on Persic et al. (1997, Gene187:9-18). Both vectors use an IgG secretory leader containing a uniquerestriction site (BssHII) to clone VH and VL at their 5′ end.Heavy-chain vector incorporates an adjacent VH linker containing aBstEII site, which is conserved across all VH subgroups, for cloning VHat the 3′ end. Thus, VH from Fab-17G05 was generated by PCR and insertedinto digested pAID4 human IgG1 heavy-chain vector (BssHII 5′ and BstEII3′). To accommodate the full range of VL subgroups at their 3′ boundary,light-chain vector incorporates a VL linker containing a XhoI site,which is available in some VL subgroups, followed by a short introncontaining a PacI site, which can be used for cloning all other VLsubgroups. Thus, VL from Fab-17G05 was generated by PCR and insertedinto digested pAID4 human kappa light-chain vector (BssHII 5′ and PacI3′). The completed constructs underwent confirmatory sequencing and weretransiently cotransfected into COS cells. COS conditioned medium wasanalyzed by Western blot to confirm antibody size and by ELISA for humanFc domain to determine antibody concentration. Antibody was alsoproduced in stably transfected CHO cells. Purification of antibodyprotein from COS or CHO cell conditioned medium was achieved by proteinA chromatography (e.g. MabSelect SuRe™, General Electric, Piscataway,N.J.), dialysis, viral filtration, and buffer exchange. The N-terminusof purified VH protein was confirmed by N-terminal sequencing to beEVQLLESGGG (SEQ ID NO: 43).

Example 7 Characterization of Ab-17G05 Binding to ActRIIB

BIACORE™-based analysis was used to characterize binding of Ab-17G05 toActRIIB. For this analysis, Ab-17G05 was purified fromCOS-cell-conditioned media by one-step protein G chromatography. FIG. 2shows kinetic characterization of Ab-17G05 binding to hActRIIB-mFc(dimeric protein), and binding parameters are listed below compared withthose of Fab-17G05.

25° C. 37° C. k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) k_(a) (M⁻¹s⁻¹) k_(d)(s⁻¹) K_(D) (M) Ab-17G05 9.7 × 10⁵ 2.7 × 10⁻⁵ 2.8 × 10⁻¹¹ 1.5 × 10⁶ 1.4× 10⁻⁴  9.2 × 10⁻¹¹ Fab-17G05  16 × 10⁵  87 × 10⁻⁵  55 × 10⁻¹¹ 2.6 × 10⁶ 31 × 10⁻⁴ 190 × 10⁻¹¹Conversion of Fab-17G05 to an antibody resulted in improvements of20-fold or greater in off-rate and K_(D) at both 25° C. or 37° C.

Example 8 Neutralization by Ab-17G05 in a Cell-Based Assay

Ab-17G05 was evaluated for its ability to neutralize binding of activinA and ActRIIB in the cell-based reporter gene assay described in Example4. In this assay, ligand-mediated activation of endogenous ActRIIA willoccur despite the presence of exogenous ActRIIB-Fc if the anti-ActRIIBantibody or Fab is neutralizing. Results obtained for Ab-R17G05 arebased on conditioned media from COS cells (quantitated by ELISA) and forFab-R17G05 on material purified with protein A. As shown in FIG. 3,Ab-17G05 was a potent stimulator of activin A signaling through ActRIIA,thus indicating that Ab-17G05 can neutralize ActRIIB binding to activinA in a cell-based system. Unconverted Fab-17G05 also displayedneutralizing capability; however, the potency of Ab-17G05 (IC50≈0.04 nM)was two orders of magnitude higher than that of Fab-17G05 (IC50≈2.6 nM).

Taken together, the foregoing findings demonstrate the generation of anantibody (17G05) capable of binding to ActRIIB with high affinity andpotently neutralizing ActRIIB-mediated signaling. Consistent with thesefindings, Ab-17G05 increases muscle mass in vivo.

Example 9 Detection of Human Anti-ActRIIB Antibodies in Serum

In the course of clinical development of an ActRIIB-Fc fusion protein(known as ACE-031), an ELISA method has been developed to detectneutralizing antibodies to the ActRIIB portion of ActRIIB-Fc in humanserum. Briefly, ACE-031 is coated on the microplate followed by controlantibody (murine or human anti-ActRIIB; 17G05) and sample incubation.After washing, samples are incubated with biotinylated Activin A andbound ligand is detected with streptavidin horseradish peroxidase (HRP)and tetramethylbenzidine (TMB) substrate. Biotinylated-Activin A bindingto ACE-031 in the absence of neutralizing antibody is recorded as maxsignal, the difference between the max signal and signal obtained in thepresence of bound neutralizing antibody (inhibition) is proportional tothe amount of neutralization activity against ACE-031 in the sample.Serum samples from human patients treated with ActRIIB-Fc and suspectedof having an immune reaction to the ActRIIB-Fc may be evaluated by thesame protocol, replacing the control antibody with the serum sample andcomparing the measured signal against the standard values generated withthe control antibody.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1-8. (canceled)
 9. An isolated polynucleotide sequence encoding ananti-ActRIIB antibody or functional fragment thereof that binds toActRIIB between amino acids 20-134 of SEQ ID NO: 1, wherein saidantibody or functional fragment thereof comprises a variable heavy chainencoded by the nucleic acid sequence of SEQ ID NO: 17 and a variablelight chain encoded by the nucleic acid sequence of SEQ ID NO:
 18. 10. Acloning or expression vector comprising the isolated polynucleotidesequence according to claim
 9. 11. An isolated host cell comprising thevector according to claim
 10. 12. A process for the production of anantibody or functional fragment thereof that binds to ActRIIB betweenamino acids 20-134 of SEQ ID NO: 1, comprising culturing the host cellof claim 11 and isolating the antibody or functional fragment thereof.13-19. (canceled)